Methods and compositions for modulating and detecting WISP activity

ABSTRACT

Methods and compositions for use in modulating the activity(s) of WISP-1 polypeptide are provided. WISP-1 antagonists include anti-WISP-1 antibodies, WISP-1 immunoadhesins and WISP-1 variants (and fusion proteins thereof) which inhibit or neutralize induction or secretion of HAS2, HA, CD44 or RHAMM by native human WISP-1 polypeptide in at least one type of mammalian cell. The invention also provides methods for in vitro, in situ, and/or in vivo diagnosis and/or treatment of mammalian cells or pathological conditions associated with native WISP-1 polypeptides.

RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.application Ser. No. 10/519,621, now pending, to which priority isclaimed and the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to methods and compositions foruse in modulating the activity(s) of WISP polypeptides, particularly,WISP-1 polypeptides. The invention also relates to methods andcompositions for in vitro, in situ, and/or in vivo diagnosis and/ortreatment of mammalian cells or pathological conditions associated withWISP polypeptides.

BACKGROUND OF THE INVENTION

Connective tissue growth factor (CTGF) is a growth factor induced infibroblasts by many factors, including TGF-β, and is essential for theability of TGF-β to induce anchorage-independent growth (AIG), aproperty of transformed cells. CTGF also is mitogenic and chemotacticfor cells, and hence growth factors in this family are believed to playa role in the normal development, growth, and repair of human tissue.Five proteins related to CTGF, including Cyr61, Nov, WISP-1, WISP2, andWISP-3 have been isolated, cloned, sequenced, and characterized asbelonging to the CCN gene family. Oemar and Luescher, Arterioscler.Thromb. Vasc. Biol., 17: 1483-1489 (1997); Brigstock, Endocrine Rev.,20:189-206 (1999). The gene encoding Cyr61 has been found to promoteangiogenesis, tumor growth, and vascularization. Babic et al., Proc.Natl. Acad. Sci. USA, 95: 6355-6360 (1998). The nov gene is expressed inthe kidney essentially at the embryonic stage, and alterations of novexpression, relative to the normal kidney, have been detected in bothavian nephroblastomas and human Wilms' tumors. Martinerie et al.,Oncogene, 9: 2729-2732 (1994). Wt1 down-regulates human nov expression,which downregulation might represent a key element in normal and tumoralnephrogenesis. Martinerie et al., Oncogene, 12: 1479-1492 (1996).

The different members of the CCN family interact with various soluble ormatrix associated macromolecules in particular sulfated glycoconjugates(Bork, FEBS Letters, 327:125-130). This interaction was used to purifyCyr61 and CTGF by affinity chromatography on heparin-agarose (Frazier etal., J. Invest. Dermatol., 107:404-411 (1996); Kireeva et al., Mol.Cell. Biol., 16:1326-1334 (1996)). Cyr61 is secreted and associated withboth the extracellular matrix and the cell surface due to its affinityfor heparan sulfate (Yang et al., Cell. Growth Diff., 2:351-357 (1991)).Recently, WISP-1 was shown to interact with decorin and biglycan, twosecreted dermatan sulfate proteoglycans. (Desnoyers, et al., J. Biol.Chem., 276:47599-47607 (2001)).

The murine protein ELM1 was recently identified in low metastatic cells.Hashimoto et al., J. Exp. Med., 187:289-296 (1998). The elm1 gene, amouse orthologue of WISP-1 disclosed below, is another member of the CNNgene family. It suppresses in vivo tumor growth and metastasis of K-1735murine melanoma cells. Another recent article on rCop-1, the ratorthologue of WISP-2 described below, describes the loss of expressionof this gene after cell transformation. Zhang et al., Mol. Cell. Biol.,18:6131-6141 (1998).

Wnts are encoded by a large gene family whose members have been found inround worms, insects, cartilaginous fish, and vertebrates. Holland etal., Dev. Suppl., 125-133 (1994). Wnts are thought to function in avariety of developmental and physiological processes since many diversespecies have multiple conserved Wnt genes. McMahon, Trends Genet., 8:236-242 (1992); Nusse and Varmus, Cell, 69: 1073-1087 (1992). Wnt genesencode secreted glycoproteins that are thought to function as paracrineor autocrine signals active in several primitive cell types. McMahon,supra (1992); Nusse and Varmus, supra (1992). The Wnt growth factorfamily includes more than ten genes identified in the mouse (Wnt-1, -2,-3A, -3B, -4, -5A, -5B, -6, -7A, -7B, -8A, -8B, -10B, -11, -12, and -13)(see, e.g., Gavin et al., Genes Dev., 4: 2319-2332 (1990); Lee et al.,Proc. Natl. Acad. Sci. USA, 92: 2268-2272 (1995); Christiansen et al.,Mech. Dev., 51: 341-350 (1995)) and at least nine genes identified inthe human (Wnt-1, -2, -3, -5A, -7A, -7B, -8B, -10B, and -11) by cDNAcloning. See, e.g., Vant Veer et al., Mol. Cell. Biol., 4: 2532-2534(1984).

The Wnt-1 proto-oncogene (int-1) was originally identified from mammarytumors induced by mouse mammary tumor virus (MMTV) due to an insertionof viral DNA sequence. Nusse and Varmus, Cell, 31: 99-109 (1982). Inadult mice, the expression level of Wnt-1 mRNA is detected only in thetestis during later stages of sperm development. Wnt-1 protein is about42 KDa and contains an amino-terminal hydrophobic region, which mayfunction as a signal sequence for secretion (Nusse and Varmus, supra,1992). The expression of Wnt-2 is detected in mouse fetal and adulttissues and its distribution does not overlap with the expressionpattern for Wnt-1. Wnt-3 is associated with mouse mammary tumorigenesis.The expression of Wnt-3 in mouse embryos is detected in the neural tubesand in the limb buds. Wnt-5a transcripts are detected in the developingfore- and hind limbs at 9.5 through 14.5 days and highest levels areconcentrated in apical ectoderm at the distal tip of limbs. Nusse andVarmus, supra (1992). Recently, a Wnt growth factor, termed Wnt-x, wasdescribed (WO95/17416) along with the detection of Wnt-x expression inbone tissues and in bone-derived cells. Also described was the role ofWnt-x in the maintenance of mature osteoblasts and the use of the Wnt-xgrowth factor as a therapeutic agent or in the development of othertherapeutic agents to treat bone-related diseases.

Wnts may play a role in local cell signaling. Peifer and Polakis,Science, 287:1606-1609 (2000). Biochemical studies have shown that muchof the secreted Wnt protein can be found associated with the cellsurface or extracellular matrix rather than freely diffusible in themedium. Papkoff and Schryver, Mol. Cell. Biol., 10: 2723-2730 (1990);Bradley and Brown, EMBO J., 9: 1569-1575 (1990).

Studies of mutations in Wnt genes have indicated a role for Wnts ingrowth control and tissue patterning. In Drosophila, wingless (wg)encodes a Wnt-related gene (Rijsewik et al., Cell, 50: 649-657 (1987))and wg mutations alter the pattern of embryonic ectoderm, neurogenesis,and imaginal disc outgrowth. Morata and Lawerence, Dev. Biol., 56:227-240 (1977); Baker, Dev. Biol., 125: 96-108 (1988); Klingensmith andNusse, Dev. Biol., 166: 396-414 (1994). In Caenorhabditis elegans,lin-44 encodes a Wnt homolog which is required for asymmetric celldivisions. Herman and Horvitz, Development, 120: 1035-1047 (1994).Knock-out mutations in mice have shown Wnts to be essential for braindevelopment (McMahon and Bradley, Cell, 62: 1073-1085 (1990); Thomas andCappechi, Nature, 346: 847-850 (1990)), and the outgrowth of embryonicprimordia for kidney (Stark et al., Nature, 372: 679-683 (1994)), tailbud (Takada et al., Genes Dev., 8: 174-189 (1994)), and limb bud. Parrand McMahon, Nature, 374: 350-353 (1995). Overexpression of Wnt-1 in themammary gland can result in mammary hyperplasia (McMahon, supra (1992);Nusse and Varmus, supra (1992)), precocious alveolar development(Bradbury et al., Dev. Biol., 170: 553-563 (1995)), and mammaryadenocarcinomas (Li et al., Oncogene, 19:1002-1009 (2000)).

Wnt-5a and Wnt-5b are expressed in the posterior and lateral mesodermand the extraembryonic mesoderm of the day 7-8 murine embryo. Gavin etal., supra (1990). These embryonic domains contribute to the AGM regionand yolk sac tissues from which multipotent hematopoietic precursors andHSCs are derived. Dzierzak and Medvinsky, Trends Genet., 11: 359-366(1995); Zon et al., in Gluckman and Coulombel, ed., Colloque, INSERM,235: 17-22 (1995), presented at the Joint International Workshop onFoetal and Neonatal Hematopoiesis and Mechanism of Bone Marrow Failure,Paris France, Apr. 3-6, 1995; Kanatsu and Nishikawa, Development, 122:823-830 (1996). Wnt-5a, Wnt-10b, and other Wnts have been detected inlimb buds, indicating possible roles in the development and patterningof the early bone microenvironment as shown for Wnt-7b. Gavin et al.,supra (1990); Christiansen et al., Mech. Devel., 51: 341-350 (1995);Parr and McMahon, supra (1995).

For a review on Wnt, see Cadigan and Nusse, Genes & Dev., 11: 3286-3305(1997).

Pennica et al., Proc. Natl. Acad. Sci., 95:14717-14722 (1998) describethe cloning and characterization of two genes, WISP-1 and WISP-2, thatare up-regulated in the mouse mammary epithelial cell line C57MGtransformed by Wnt-1, and a third related gene, WISP-3. (See also, WO99/21998 published May 6, 1999; WO 99/21999 published May 6, 1999).Pennica et al. report that these WISP genes may be downstream of Wnt-1signaling and that aberrant levels of WISP expression in colon cancermay play a role in colon tumorigenesis. WISP-1 has recently beenidentified as a β-catenin-regulated gene and the characterization of itsoncogenic activity demonstrated that WISP-1 might contribute toβ-catenin-mediated tumorigenesis (Xu et al., Gene & Develop., 14:585-595(2000)). WISP-1 overexpression in normal rat kidney cells (NRK-49F)induced morphological transformation, accelerated cell growth andenhanced saturation density. In addition, these cells readily formtumors when injected into nude mice suggesting that WISP-1 may play somerole in tumorigenesis (Xu et al., supra 2000). WISP-1 is alsooverexpressed in transformed human breast cancer cell lines and in about47% of primary human breast cancer associated with certain advancedfeatures. Xie et al., Cancer Res., 61:8917-8923 (2001); Saxena et al.,Mol. Cell Biochem., 228:99-104 (2001); Michaelson et al., Oncogene,20:5093-5099 (2001). A particular WISP-1 variant has also been reportedto be overexpressed in about 86% of human scirrhous gastric carcinomacells. Tanaka et al., Oncogene, 20:5525-5532 (2001).

Hurvitz et al., Nature Genetics, 23:94-97 (1999) describe a studyinvolving WISP3 in which nine different mutations of WISP3 in unrelatedindividuals were found to be associated with the autosomal recessiveskeletal disorder, progressive pseudorheumatoid dysplasia (PPD). WISP3expression by RT-PCR was observed by Hurvitz et al. in humansynoviocytes, articular cartilage chondrocytes, and bone-marrow-derivedmesenchymal progenitor cells.

PCT application WO98/21236 published May 22, 1998 discloses a humanconnective tissue growth factor gene-3 (CTGF-3) encoding a 26 kD memberof the growth factor superfamily. WO98/21236 discloses that the CTGF-3amino acid sequence was deduced from a human osteoblast cDNA clone, andthat CTGF-3 was expressed in multiple tissues like ovary, testis, heart,lung, skeletal muscle, adrenal medulla, adrenal cortex, thymus,prostate, small intestine and colon.

Hyaluronic acid (also referred to as HA, hyaluronate, or hyaluronan) isrecognized in the literature as being an important component of theextracellular matrix (See, e.g., Hardingham et al., FASEB J., 6:861-870(1992); Laurent et al., FASEB J., 6:2397-2404 (1992)). HA is a componentof skin and mesenchymal tissues where it facilitates cell migrationduring wound healing, inflamation, and embryonic morphogenesis. (Knudsonet al., FASEB J., 7:1233-1241 (1993); Knudson et al., CIBA Found. Symp.,143:150-169 (1989)). HA has also been reported to play a role in certaintypes of metastases. (Naor et al., CD44: Structure, Function andAssociation with the Malignant Process, Advances in Cancer Research,Academic Press (1997), pages 241-319). The largest concentrations of HAare found in the skin and musculo-skeletal system which account for over50% of total body HA. (Banerji et al., J. Cell Biol., 144:789-801(1999)).

Various investigators have reported on receptors which bind HA. One ofthe receptors identified for HA is the CD44 protein. (See, e.g., Cultyet al., J. Cell Biology, 111:2765-2774 (1990); Aruffo et al., Cell,61:1303-1313 (1990); Naor et al., CD44: Structure, Function andAssociation with the Malignant Process, Advances in Cancer Research,Academic Press (1997), pages 241-319); Ropponen et al., Cancer Res.,58:342-347 (1998); Masaki et al., Cancer, 92:2539-2546 (2001). CD44 is afamily of cell-surface glycoproteins generated from a single gene byalternative splicing and differential glycosylation. (Wielenga et al.,Am. J. Pathology, 154:515-523 (1999)). CD44 is believed to function as acell adhesion receptor, linking extracellular molecules, specificallyhyaluronate, to the cell and the cytoskeleton (Wielenga et al., supra).CD44 is expressed on epithelial, mesenchymal and lymphoid cells. (Lesleyet al., Adv. Immunol., 54:271-335 (1994)). Wielenga et al. report thatCD44 expression may be regulated by the WNT pathway, based on certainexperiments analyzing CD44 expression in the intestinal mucosa of miceand humans with genetic defects in either APC or Tcf-4. (Wielenga etal., supra).

Other HA receptors characterized to date include RHAMM (also referred toas receptor for hyaluronic acid mediated motility), a 58 kDintracellular protein expressed transiently on the surface oftransformed lymphocytes (Hardwick et al., J. Cell Biol., 117:1343-1350(1992); Turley et al., Exp. Cell Res., 207:277-282 (1993)). RHAMMexpression in fibroblasts was reported to promote metastasis and play animportant role in H-Ras transformation (Hall et al., infra).

Another receptor which binds HA was described by Banerji et al. (Banerjiet al., supra). Banerji et al. report a receptor on lymph vessel walls,referred to as “LYVE-1”, which is a 322-residue type I integral membranepolypeptide which has a 41% similarity to the CD44 receptor. Unlike theCD44 receptor for HA, the LYVE-1 protein is absent in blood vessels. Inaddition, layilin (Bono et al., Mol. Biol. Cell, 12:891-900 (2001)) andHARE (Zhou et al., J. Biol. Chem., 275:37733-37741 (2000)) were alsodescribed as HA receptors.

SUMMARY OF THE INVENTION

Applicant has surprisingly found that WISP-1 can induce HAS2 (hyaluronansynthase 2), CD44, and RHAMM mRNA expression, CD44 protein synthesis,and HA secretion. The induction or secretion of such molecules maypromote or increase cancer cell growth, motility and/or metastaticpotential. The present invention thus provides for instance, WISP-1antagonists and methods of using such antagonists. The antagonistsdescribed herein find utility for, among other things, in vitro, insitu, or in vivo diagnosis or treatment of mammalian cancer cells orother pathological conditions associated with the induction or secretionof HAS2, HA, CD44 or RHAMM.

In embodiments of the invention, there are provided isolated WISP-1antagonists. Such antagonists may comprise antibodies, such as WISP-1antibodies. In preferred embodiments, the antagonists may block orneutralize WISP-1 induction or secretion of HAS2, HA, CD44 or RHAMM.Such antagonistic antibodies may, for example, be monoclonal antibodies,chimeric antibodies, humanized antibodies, or human antibodies. TheWISP-1 antagonists contemplated for use in the invention include WISP-1immunoadhesins, WISP-1 variants, covalently modified forms thereof, orfusion proteins thereof. By way of example, such antagonists may includepegylated WISP-1 or WISP-1 fused to heterologous sequences such asepitope tags or leucine zippers. The methods contemplate the use of asingle type of antagonist molecule or a combination of two or more typesof antagonists.

The methods of the invention include methods to treat pathologicalconditions or diseases in mammals associated with or resulting fromWISP-1, including, induction or secretion of HAS2, HA, CD44 or RHAMM byWISP-1. In the methods of treatment, WISP-1 antagonists may beadministered to the mammal suffering from such pathological condition ordisease. For example, the invention provides a method comprisingexposing a mammalian cell(s), such as a cancer cell(s), to one or moreWISP-1 antagonists in an amount effective to decrease, neutralize orblock WISP-1 induction or secretion of HAS2, HA, CD44 or RHAMM. The cellmay be in cell culture or in a mammal, e.g. a mammal suffering from, forinstance, cancer.

The invention also provides compositions which comprise one or moreWISP-1 antagonists. Optionally, the compositions of the invention willinclude pharmaceutically acceptable carriers or diluents. Preferably,the compositions will include one or more WISP-1 antagonists in anamount which is therapeutically effective to treat a pathologicalcondition or disease.

The invention also provides articles of manufacture and kits whichinclude one or more WISP-1 antagonists.

The invention also provides methods of conducting screening assays toidentify candidate molecules, such as small molecule compounds,polypeptides or antibodies, which act as antagonists with respect toblocking or neutralizing WISP-1 induction or secretion of HAS2, HA, CD44or RHAMM.

More particular embodiments of the invention include isolated WISP-1antagonists which inhibit or neutralize the induction or secretion ofHAS2, HA, CD44 or RHAMM by native WISP-1 polypeptide in at least onetype of mammalian cell, said antagonist being selected from the groupconsisting of an anti-WISP-1 antibody, a WISP-1 immunoadhesin, a WISP-1variant, and fusion proteins thereof. The antagonist may comprise ananti-WISP-1 antibody which binds native human WISP-1 polypeptidecomprising amino acids 23-367 of FIGS. 9A-9C or one or more domains ofWISP-1 comprising amino acids encoded by the sequences of SEQ ID NO:3;4; 5; 6; 7; 8; 9; 10; or 11 herein. The anti-WISP-1 antibody may be achimeric, humanized or human antibody.

The invention also provides compositions comprising the antagonistsdescribed herein and a carrier, optionally the carrier is apharmaceutically-acceptable carrier.

The invention also provides methods of inhibiting or neutralizing WISP-1induction or secretion of HAS2, HA, CD44 or RHAMM in mammalian cells,comprising exposing said mammalian cells to an effective amount ofWISP-1 antagonist, wherein said WISP-1 antagonist is selected from thegroup consisting of

-   a) a WISP-1 immunoadhesin;-   b) a WISP-1 polypeptide linked to a nonproteinaceous polymer    selected from the group consisting of polyethylene glycol,    polypropylene glycol, and polyoxyalkylene;-   c) a WISP-1 antibody; and-   d) a WISP-1 variant.    The WISP-1 immunoadhesins employed in the methods may comprise a    WISP-1 sequence fused to a Fc region of an immunoglobulin. The    anti-WISP-1 antibodies employed in the methods may bind native human    WISP-1 comprising amino acids 23-367 of FIGS. 9A-9C, or one or more    of the domains of WISP-1 described in the Examples below. In the    methods, the mammalian cells may comprise cancer cells, and    optionally the cells comprise colon or colorectal cancer cells,    breast cancer cells, lung cancer cells, or brain cancer cells (such    as glioma or glioblastoma).

In other embodiments, there are provided methods of treating cancer in amammal, comprising administering to said mammal an effective amount ofWISP-1 antagonist. Optionally, in said methods, the antagonist mayinhibit or neutralize induction or secretion of HAS2, HA, CD44 or RHAMMby native human WISP-1 polypeptide in at least one type of mammaliancell and said antagonist is selected from the group consisting of ananti-WISP-1 antibody, a WISP-1 immunoadhesin and a WISP-1 variant.Optionally, the antagonist may act to inhibit cancer cell growth orcancer cell metastasis. The cancer in the mammal may comprise colon orcolorectal cancer cells, breast cancer cells, lung cancer cells, orbrain cancer cells (such as glioma or glioblastoma). Optionally, theantagonist(s) employed in the methods inhibits or reduces cancer cellgrowth or metastasis. Also, in the methods, chemotherapy, radiation,proodrug, cytotoxic agent, growth inhibitory agent, or cytokine may alsobe administered to the mammal.

In more particular embodiments, there are provided antibodies whichspecifically bind to one or more domains of WISP-1 polypeptide(described further in the Examples below) comprising amino acids encodedby the sequences of SEQ ID NO:3; 4; 5; 6; 7; 8; 9; 10; or 11 herein.Optionally, the antibody is a monoclonal antibody. Optionally, themonoclonal antibody comprises the 3D11, 11C2, 9C10, 5D4, or 9C11antibody secreted by the hybridoma deposited with ATCC as accessionnumber PTA-4624, PTA-4628, PTA-4626, PTA-4625, or PTA-4627,respectively.

Also provided are antibodies which bind to the same epitope as theepitope to which the 3D11, 11C2, 9C10, 5D4, or 9C11 monoclonal antibodyproduced by the hybridoma cell line deposited as ATCC accession numberPTA-4624, PTA-4628, PTA-4626, PTA-4625, or PTA-4627, respectively,binds.

In yet other particular embodiments, there is provided the hybridomacell line which produces monoclonal antibody 3D11, 11C2, 9C10, 5D4, or9C11 and deposited with ATCC as accession number PTA-4624, PTA-4628,PTA-4626, PTA-4625, or PTA-4627, respectively, and the monoclonalantibody 3D11, 11C2, 9C10, 5D4, or 9C11 secreted by the hybridomadeposited with ATCC as accession number PTA-4624, PTA-4628, PTA-4626,PTA-4625, or PTA-4627, respectively.

There are also provided isolated anti-WISP-1 monoclonal antibodies,comprising antibodies which bind to WISP-1 polypeptide and competitivelyinhibit binding of the monoclonal antibody produced by the hybridomadeposited as ATCC accession no. PTA-4624, PTA-4628, PTA-4626, PTA-4625,or PTA-4627 to said WISP-1 polypeptide.

There are also provided chimeric or humanized anti-WISP-1 antibodieswhich specifically bind to WISP-1 polypeptide and comprise (a) asequence derived from the 3D11, 11C2, 9C10, 5D4, or 9C11 antibodysecreted by the hybridoma deposited with ATCC as accession numberPTA-4624, PTA-4628, PTA-4626, PTA-4625, or PTA-4627, respectively.Optionally, such antibodies may comprise a heavy chain, light chain orvariable regions derived from the 3D11, 11C2, 9C10, 5D4, or 9C11antibody.

The anti-WISP-1 antibodies may be linked to one or morenon-proteinaceous polymers selected from the group consisting ofpolyethylene glycol, polypropylene glycol, and polyoxyalkylene, or to acytotoxic agent or enzyme, or to a radioisotope, fluorescent compound orchemiluminescent compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F show WISP-1 promotion of hyaluronan production. Particleexclusion assay demonstrating a hyaluronan coat at the surface ofNRK/WISP-1H (A) and NRK/WISP-1L (B) but absent from NRK/control (C).NRK/WISP-1H (D) and NRK/control (E) were stained for hyaluronan withbHABP. F, Time course of HA accumulation in NRK/WISP-1H and NRK/controlmedia.

FIGS. 2A-2C show WISP-1 increases HAS2, CD44 and RHAMM mRNA expressionand CD44 protein expression. A, Real time RT-PCR analysis of HAS1, HAS2,HAS3, CD44, RHAMM and hyaluronidase (Hyal) mRNA expression inNRK/WISP-1H, NRK/WISP-1L and NRK/control cells. Results are given asfold induction over the expression in NRK/control cells. B, Flowcytometry analysis of CD44 expression in NRK/WISP-1H and NRK/controlcells. The shaded area represents the fluorescence intensity of thesecondary antibody alone. C, Western blot analysis of CD44 protein inNRK/WISP-1H and NRK/control cells. Actin staining was used as a loadingcontrol.

FIGS. 3A-3G show WISP-1 expression increases cell motility and modifiescellular morphology. NRK/control cells (A) formed defined colonies whenplated at low density whereas NRK/WISP-1L (B) and NRK/WISP-1H cells (C)scattered. NRK/WISP-1H (E) showed a de-differentiated spindloidmorphology with lamelipodia compared to NRK/control cells (D). F, Therandom migration of NRK/WISP-1H and NRK/control cells was measured over15 hours using time lapse microscopy. Results represent the typicalaverage migration distance of cells in one field. G, Motility ofNRK/WISP-1H and NRK/control cells was evaluated by a cell wound healingassay and measured after 15 hours using time lapse microscopy. Datarepresent the results of a typical experiment.

FIGS. 4A-4C show WISP-1 addition induces HAS2 mRNA expression andhaptotactic migration of NRK cells. A, Real time RT-PCR analysis of HAS2and CD44 expression in NRK cells seeded on coated surface. In certaincases the coated substrates were further incubated with WISP-1(treatment). Results are given in fold induction over expression in NRKcells plated on non-coated, non-treated surface. The underside ofmodified Boyden chamber filters were coated and NRK cells (B) or SW480cells (C) were added to the upper chamber. In certain cases, additionswere made directly to the lower chamber. Cell motility was evaluated bycounting the cells that migrated to the lower side of the insert.

FIGS. 5A-5E show WISP-1, HAS2 and CD44 over-expression and increasedcell motility in C57MG/Wnt-1 cells and MMTV-Wnt-1 transgenic micemammary tumors. A, Real time RT-PCR analysis of WISP-1, HAS2 and CD44expression in C57MG/Wnt-1 and C57MG/control cells. Results are given asfold induction over expression in C57MG/control cells. B, Western blotanalysis of CD44 content in C57MG/Wnt-1 and C57MG/control cells. Actinstaining was used as a loading control. C, C57MG/control cells formeddistinct colonies when plated at low density whereas C57MG/Wnt-1 cellsscattered. Semi-quantitative RT-PCR analysis of HAS2 (D) and CD44 (E) inmammary tumors of MMTV-Wnt-1 transgenic mice. Results are expressed asrelative fold induction over the expression in normal mammary glandtissue.

FIGS. 6A-6H show WISP-1, HAS2 and CD44 expression and HA accumulation inMMTV-Wnt-1 transgenic mammary tumors. In situ hybridization of WISP-1(A, B) HAS2 (C, D) and CD44 (E, F), immunohistochemistry of CD44 (G) andbHABP fluorescent staining of hyaluronan (H) in MMTV-Wnt-1 transgenicmouse mammary tumors. t, tumor; s, stroma.

FIGS. 7A-7L show WISP-1 expression promotes metastatic lung colonizationand tumor growth. The effect of WISP-1 was analyzed by injectingNRK/control, NRK/WISP-1L or NRK/WISP-1H cells in the tail vein of nudemice. At various times after injection, lungs were imaged by MRI (A, D,G, J), excised and their gross appearance (B, E, H, K) and histologicalfeatures (C, F, I, L) were recorded. The severity of lung invasion bytumors was graded as normal (A, B, C); grade I (D, E, F); grade ll (G,H, I) or grade III (J, K, L).

FIGS. 8A-B show CD44 antibody prevents NRK/WISP-1H cell metastasis.NRK/WISP-1H cells (2.5×10⁵ cells) were injected in the tail vein of nudemice. Mice were treated twice a week with 10 mg/kg of a CD44 antibody,an isotype control antibody or with buffer only (PBS). The lungs wereexcised after four weeks for gross anatomical analysis. Picture of anormal lung is shown for comparison.

FIGS. 9A-9C show the nucleotide (SEQ ID NO:2) and putative amino acidsequence (SEQ ID NO:1) of native human WISP-1.

FIG. 10 shows the effects of WISP-1 expression on NRK metastaticpotential.

FIGS. 11A-B show the binding properties of WISP-1 antibodies to variousWISP-1 constructs and preparations.

FIGS. 12A-G show diagrams of various domains of WISP-1 polypeptide andthe results of assays conducted to identify the epitopes recognized bythe WISP-1 antibodies 11C2, 5D4, 9C11, and 3D11, respectively.

FIG. 13 shows the results on an assay conducted to identify the epitoperecognized by the WISP-1 antibody 9C10.

FIG. 14 shows the results of an ELISA demonstrating detection of WISP-1by WISP-1 antibodies.

FIG. 15 shows the results of an ELISA binding assay demonstrating thatWISP-1 antibodies recognizing the variable region of WISP-1 can inhibitWISP-1 binding to heparin.

FIGS. 16A-B show the results of an assay detecting inhibition ofhaptotaxis of NRK cells by WISP-1 antibodies (16A) and a chartsummarizing the properties and characteristics of WISP-1 antibodies(16B).

FIGS. 17A-E show the results of an in vivo study of the effects ofWISP-1 antibodies. After 3 weeks, the severity of the lesions found inWISP-1 antibody treated animals was greatly attenuated compared tocontrol (FIG. 17 a, b). The number of nodules and the average area ofthe metastatic foci found in mice treated with WISP-1 antibodies (n=5)were reduced compared to animals treated with a control antibody (FIG.17 c,d). The total pulmonary area covered by the lesions was reduced by82-97% compared to animal treated with an isotype control antibody (FIG.17 e).

FIG. 18 is a bar diagram showing expression of WISP-1 in 4T1/control,4T1/WISP-1L, 4T1/WISP-1H, NRK/control, NRK/WISP-1L, and WISP-1H celllines as measured by semi-quantitative RT-PCR (Taqman).

FIG. 19 shows the results of an in vitro assay on colony formation of4T1/control, 4T1/WISP-1L, and 4T1/WISP-1H cell lines.

FIG. 20 shows the results of an in vitro assay measuring invasion indexusing a Matrigel modified Boyden chamber system and 4T1/control,4T1/WISP-1L, and 4T1/WISP-1H cell lines.

FIGS. 21A-B show effects of WISP-1 expression on mammary epithelial celltumorigenesis. The effect of WISP-1 was analyzed by injecting4T1/control 1, 4T1/control 2, 4T1/WISP-1L or 4T1/WISP-1H cells in Balb/Cmice. Tumor volume (21A) and tumor weight (21 B) are reported.

FIG. 22 is a bar diagram illustrating the relative expression of HAS2and CD44 in tumors formed by the inoculated 4T1/control, 4T1/WISP-1L and4T1/WISP-1H cells.

FIGS. 23A-F illustrate the WISP-1 effects on mammary epithelial cellsmetastasis evaluated in vivo by inoculating 4T1 cells in mice mammaryfat pads and examining the extent of the metastatic propagation by microcomputer tomography and histology. After 31 days, the mice inoculatedwith 4T1/WISP-1L or 4T1/WISP-1H cells had extensive lung metastasis(FIGS. 23 b and 23 d) compared to the 4T1/control injected mice (FIGS.23 a and 23 c). Using immunohistochemistry, it was also observed thatthe 4T1/WISP-1 pulmonary metastatic foci expressed high levels of CD44(FIG. 23 e). In these tumors, CD44 was localized at the plasma membraneof the 4T1/WISP-1 cells (FIG. 23 f).

FIG. 24A shows WISP-1 expression in NRK/control, NRK/WISP-1_(—)1234,NRK/WISP-1_(—)134, NRK/WISP-1_(—)234, NRK/WISP-1_(—)123 andNRK/WISP-1_(—)124 cells lines, as measured by semi-quantitative RT-PCR(Taqman); the results are expressed as fold relative to WISP-1expression in NRK/WISP-1_(—)1234. FIG. 24B illustrates the effects ofthese constructs on tumorigenesis in vivo; the results are shown by theeffects on tumor volume.

FIG. 25 shows the histological analysis of the excised tumors referredto in FIG. 24 and revealed that the neoplastic cells fromNRK/WISP-1_(—)234 tumors were phenotypically similar to the neoplasticcells from NRK/WISP-1_(—)1234 tumors. In these tumors, cells appearedfibroblastic, differentiated and spindloid. The neoplastic cells fromNRK/WISP-1_(—)134 tumors were phenotypically different and appeared lessdifferentiated, frequently multinucleated and had a brisk mitotic rateas demonstrated by the presence of multiple mitotic figures (arrows).

FIG. 26 shows the histological analysis of the excised tumors in theNRK/WISP-1_(—)134 xenograft referred to in FIG. 24, and reveals theneoplastic cells invaded several blood vessels adjacent to the tumor.

FIG. 27 shows expression as fold invasion compared to a control in whichHEK 293 cells transfected with an empty vector were plated in themodified Boyden chamber system. 4T1 cells demonstrated a 4 fold increaseinvasion when HEK 293 cells expressing constructs WISP-1_(—)1234,WISP-1_(—)134 or WISP-123 were seeded in the lower chamber.

FIGS. 28A-28B illustrate the effects of soluble WISP-1-domain-1 onWISP-1 promoted 4T1 cell invasion, as evaluated using a Matrigel coatedmodified Boyden chamber system. The results are expressed as relativefold invasion compared to 4T1/control cells. WISP-1-domain-1-His andWISP-1-domain-1-Fc demonstrated a dose dependent antagonistic activitytoward WISP-1 promoted 4T1 invasion.

FIG. 29 illustrates an in situ hybridization analysis of tissues fromthe HPAC xenograft model of human pancreatic adenocarcinomas to evaluateWISP-1 expression.

FIG. 30 shows a graph illustrating the effects of WISP-1 constructs ontumor volume in a nude mouse model.

FIG. 31 shows in situ hybridization analyses evaluating WISP-1expression in primary human pancreatic adenocarcinomas.

FIG. 32 shows immunohistochemical staining evaluating WISP-1 expressionin primary colon adenocarcinomas.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The term “WISP polypeptide” refers to the family of native-sequencehuman and mouse WISP proteins and variants described herein whose genesare induced at least by Wnt-1. This term includes WISP-1 and variantsthereof. Such WISP-1 proteins are described further below and in PCTapplication WO99/21998 published May 6, 1999, U.S. Pat. No. 6,387,657 B1issued May 14, 2002, and in Pennica et al., Proc. Natl. Acad. Sci.,95:14717-14722 (1998).

The terms “WISP-1 polypeptide”, “WISP-1 homologue” and grammaticalvariants thereof, as used herein, encompass native-sequence WISP-1protein and variants (which are further defined herein). The WISP-1polypeptide may be isolated from a variety of sources, such as fromhuman tissue types or from another source, or prepared by recombinant orsynthetic methods, or by any combination of these and similartechniques.

The term “native-sequence WISP-1 polypeptide” comprises a polypeptidehaving the same amino acid sequence as a WISP-1 polypeptide derived fromnature. Such native-sequence WISP-1 polypeptides can be isolated fromnature or can be produced by recombinant or synthetic means. The term“native-sequence WISP-1 polypeptide” specifically encompasses naturallyoccurring truncated or secreted forms of a WISP-1 polypeptide disclosedherein, naturally occurring variant forms (e.g., alternatively splicedforms or splice variants), and naturally occurring allelic variants of aWISP-1 polypeptide. In one embodiment of the invention, thenative-sequence WISP-1 polypeptide is a mature or full-lengthnative-sequence human WISP-1 polypeptide comprising amino acids 23 to367 of FIG. 9 or amino acids 1 to 367 of FIG. 9, respectively, with orwithout N-terminal methionine.

In another embodiment of the invention, the native-sequence WISP-1polypeptide is the full-length or mature native-sequence human WISP-1polypeptide comprising amino acids 23 to 367 or 1 to 367 of FIG. 9wherein the valine residue at position 184 or the alanine residue atposition 202 has/have been changed to an isoleucine or serine residue,respectively, with or without N-terminal methionine. In anotherembodiment of the invention, the native-sequence WISP-1 polypeptide isthe full-length or mature native-sequence human WISP-1 polypeptidecomprising amino acids 23 to 367 or 1 to 367 of the FIG. 9 wherein thevaline residue at position 184 and the alanine residue at position 202has/have been changed to an isoleucine or serine residue, respectively,with or without N-terminal methionine.

In another embodiment of the invention, the native-sequence WISP-1polypeptide is one which is encoded by a nucleotide sequence comprisingone of the human WISP-1 splice or other native-sequence variants,including SEQ ID NOS:23, 24, 25, 26, 27, 28, or 29 shown in WO99/21998,with or without an N-terminal methionine.

The term “WISP-1 variant” means an active WISP-1 polypeptide as definedbelow having at least about 80%, preferably at least about 85%, 86%,87%, 88%, 89%, more preferably at least about 90%, 91%, 92%, 93%, 94%,most preferably at least about 95%, 96%, 97%, 98%, or 99% amino acidsequence identity with human mature WISP-1 having the deduced amino acidsequence shown in FIG. 9, or a soluble fragment thereof. Such variantsinclude, for instance, WISP-1 polypeptides wherein one or more aminoacid residues are added to, or deleted from, the N- or C-terminus of thefull-length or mature sequences of FIG. 9, or WISP-1 polypeptideswherein one or more amino acid residues are inserted or deleted from theinternal sequence or domains of the polypeptide, including variants fromother species, but excludes a native-sequence WISP-1 polypeptide.Preferably such a variant acts as an antagonist, as defined below.

An “extracellular domain”, “ECD” or “secreted” protein refers to a formof a polypeptide which is essentially free of any transmembrane andcytoplasmic domains. A “secreted” form of a protein or polypeptide mayor may not include a signal sequence.

“Stringent conditions” are those that (1) employ low ionic strength andhigh temperature for washing, for example, 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; (3) employ50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS; or (4) employa buffer of 10% dextran sulfate, 2×SSC (sodium chloride/sodium citrate),and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989), and include the use of a washing solution andhybridization conditions (e.g., temperature, ionic strength, and percentSDS) less stringent than described above. An example of moderatelystringent conditions is a condition such as overnight incubation at 37°C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmonsperm DNA, followed by washing the filters in 1×SSC at about 37-50° C.The skilled artisan will recognize how to adjust the temperature, ionicstrength, etc., as necessary to accommodate factors such as probe lengthand the like.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the WISP naturalenvironment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as the insulin and insulin variants disclosed herein) to a mammal.The components of the liposome are commonly arranged in a bilayerformation, similar to the lipid arrangement of biological membranes.

The terms “amino acid” and “amino acids” refer to all naturallyoccurring L-alpha-amino acids. This definition is meant to includenorleucine, ornithine, and homocysteine. The amino acids are identifiedby either the single-letter or three-letter designations: Asp D asparticacid Ile I isoleucine Thr T threonine Leu L leucine Ser S serine Tyr Ytyrosine Glu E glutamic acid Phe F phenylalanine Pro P proline His Hhistidine Gly G glycine Lys K lysine Ala A alanine Arg R arginine Cys Ccysteine Trp W tryptophan Val V valine Gln Q glutamine Met M methionineAsn N asparagine

In the Sequence Listing and Figures, certain other single-letter orthree-letter designations may be employed to refer to and identify twoor more amino acids or nucleotides at a given position in the sequence.

“Percent (%) amino acid sequence identity” with respect to thepolypeptide sequences identified herein is defined as the percentage ofamino acid residues in a candidate sequence that are identical with theamino acid residues in such a polypeptide sequence identified herein,after aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are obtained as describedbelow by using the sequence comparison computer program ALIGN-2, whereinthe complete source code for the ALIGN-2 program is provided in thetable below. The ALIGN-2 sequence comparison computer program wasauthored by Genentech, Inc. and the source code shown in the table belowhas been filed with user documentation in the U.S. Copyright Office,Washington D.C., 20559, where it is registered under U.S. CopyrightRegistration No. TXU510087. The ALIGN-2 program is publicly availablethrough Genentech, Inc., South San Francisco, Calif. or may be compiledfrom the source code provided in the table below. The ALIGN-2 programshould be compiled for use on a UNIX operating system, preferablydigital UNIX V4.0D. All sequence comparison parameters are set by theALIGN-2 program and do not vary.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a polypeptide fused to a “tag polypeptide”. Thetag polypeptide has enough residues to provide an epitope against whichan antibody can be made. The tag polypeptide preferably also is fairlyunique so that the antibody does not substantially cross-react withother epitopes. Suitable tag polypeptides generally have at least sixamino acid residues and usually between about 8 and 50 amino acidresidues (preferably, between about 10 and 20 amino acid residues).

As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

The term “antagonist” is used in the broadest sense, and includes anymolecule that partially or fully blocks, inhibits, or neutralizes one ormore biological activities of WISP-1 polypeptide, in vitro, in situ, orin vivo. Examples of such biological activities of WISP-1 polypeptidesinclude induction or secretion of HAS2, HA, CD44 or RHAMM in at leastone type of mammalian cell. An antagonist may function in a direct orindirect manner. For instance, the antagonist may function to partiallyor fully block, inhibit or neutralize one or more biological activitiesof WISP-1 polypeptide, in vitro, in situ, or in vivo, e.g., as a resultof its direct binding to WISP-1 polypeptide. The antagonist may alsofunction indirectly to partially or fully block, inhibit or neutralizeone or more biological activities of WISP-1 polypeptide, in vitro, insitu, or in vivo as a result of, e.g., blocking or inhibiting anothereffector molecule.

The term “WISP-1 antagonist” refers to any molecule that partially orfully blocks, inhibits, or neutralizes a biological activity of WISP-1and include but are not limited to, antibodies, immunoadhesins, WISP-1immunoadhesins, WISP-1 fusion proteins, covalently modified forms ofWISP-1, WISP-1 variants and fusion proteins thereof, WISP-1 antibodies,and higher oligomer forms of WISP-1 (dimers, aggregates) or homo- orheteropolymer forms of WISP-1. To determine whether a WISP-1 antagonistmolecule partially or fully blocks, inhibits or neutralizes a biologicalactivity of WISP-1, assays may be conducted to assess the effect(s) ofthe antagonist molecule on, for example, various cells (as described inthe Examples) or in an in vivo murine model of lung cancer metastasis.The various assays may be conducted in known in vitro or in vivo assayformats. Preferably, the WISP-1 antagonists employed in the methodsdescribed herein will be capable of blocking or neutralizing at leastone type of WISP-1 activity, which may optionally be determined inassays such as described herein.

The term “antibody” is used in the broadest sense and specificallycovers, for example, single monoclonal antibodies, antibody compositionswith polyepitopic specificity, single chain antibodies, and fragments ofantibodies. “Antibody” as used herein includes intact immunoglobulin orantibody molecules, polyclonal antibodies, multispecific antibodies(i.e., bispecific antibodies formed from at least two intact antibodies)and immunoglobulin fragments (such as Fab, F(ab′)₂, or Fv), so long asthey exhibit any of the desired antagonistic properties describedherein.

Antibodies are typically proteins or polypeptides which exhibit bindingspecificity to a specific antigen. Native antibodies are usuallyheterotetrameric glycoproteins, composed of two identical light (L)chains and two identical heavy (H) chains. Typically, each light chainis linked to a heavy chain by one covalent disulfide bond, while thenumber of disulfide linkages varies between the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light and heavy chain variable domains [Chothia etal., J. Mol. Biol., 186:651-663 (1985); Novotny and Haber, Proc. Natl.Acad. Sci. USA, 82:4592-4596 (1985)]. The light chains of antibodiesfrom any vertebrate species can be assigned to one of two clearlydistinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains, immunoglobulinscan be assigned to different classes. There are five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may befurther divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3,and IgG-4; IgA-1 and IgA-2. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called alpha,delta, epsilon, gamma, and mu, respectively.

“Antibody fragments” comprise a portion of an intact antibody, generallythe antigen binding or variable region of the intact antibody. Examplesof antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments,diabodies, single chain antibody molecules, and multispecific antibodiesformed from antibody fragments.

The term “variable” is used herein to describe certain portions of thevariable domains which differ in sequence among antibodies and are usedin the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not usually evenlydistributed through the variable domains of antibodies. It is typicallyconcentrated in three segments called complementarity determiningregions (CDRs) or hypervariable regions both in the light chain and theheavy chain variable domains. The more highly conserved portions of thevariable domains are called the framework (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largelyadopting a β-sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the β-sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen binding site of antibodies [see Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, NationalInstitutes of Health, Bethesda, Md. (1987)]. The constant domains arenot involved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen.

The monoclonal antibodies herein include chimeric, hybrid andrecombinant antibodies produced by splicing a variable (includinghypervariable) domain of the antibody of interest with a constant domain(e.g. “humanized” antibodies), or a light chain with a heavy chain, or achain from one species with a chain from another species, or fusionswith heterologous proteins, regardless of species of origin orimmunoglobulin class or subclass designation, as well as antibodyfragments (e.g., Fab, F(ab′)₂, and Fv), so long as they exhibit thedesired biological activity or properties. See, e.g. U.S. Pat. No.4,816,567 and Mage et al., in Monoclonal Antibody Production Techniquesand Applications, pp. 79-97 (Marcel Dekker, Inc.: New York, 1987).

Thus, the modifier “monoclonal” indicates the character of the antibodyas being obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler and Milstein,Nature, 256:495 (1975), or may be made by recombinant DNA methods suchas described in U.S. Pat. No. 4,816,567. The “monoclonal antibodies” mayalso be isolated from phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990), for example.

“Humanized” forms of non-human (e.g. murine) antibodies are specificchimeric immunoglobulins, immunoglobulin chains, or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat, or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, the humanized antibody may comprise residues which arefound neither in the recipient antibody nor in the imported CDR orframework sequences. These modifications are made to further refine andoptimize antibody performance. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin consensus sequence.The humanized antibody optimally also will comprise at least a portionof an immunoglobulin constant region or domain (Fc), typically that of ahuman immunoglobulin.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies known inthe art or as disclosed herein. This definition of a human antibodyincludes antibodies comprising at least one human heavy chainpolypeptide or at least one human light chain polypeptide, for examplean antibody comprising murine light chain and human heavy chainpolypeptides. Human antibodies can be produced using various techniquesknown in the art. In one embodiment, the human antibody is selected froma phage library, where that phage library expresses human antibodies(Vaughan et al. Nature Biotechnology, 14:309-314 (1996): Sheets et al.PNAS, (USA) 95:6157-6162 (1998)); Hoogenboom and Winter, J. Mol. Biol.,227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Humanantibodies can also be made by introducing human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology, 10: 779-783(1992); Lonberg et al., Nature, 368: 856-859 (1994); Morrison, Nature,368:812-13 (1994); Fishwild et al., Nature Biotechnology, 14: 845-51(1996); Neuberger, Nature Biotechnology, 14: 826 (1996); Lonberg andHuszar, Intern. Rev. Immunol., 13:65-93 (1995). Alternatively, the humanantibody may be prepared via immortalization of human B lymphocytesproducing an antibody directed against a target antigen (such Blymphocytes may be recovered from an individual or may have beenimmunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,147 (1):86-95 (1991); and U.S. Pat. No. 5,750,373.

The term “Fc region” is used to define the C-terminal region of animmunoglobulin heavy chain which may be generated by papain digestion ofan intact antibody. The Fc region may be a native sequence Fc region ora variant Fc region. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue at aboutposition Cys226, or from about position Pro230, to the carboxyl-terminusof the Fc region (using herein the numbering system according to Kabatet al., supra). The Fc region of an immunoglobulin generally comprisestwo constant domains, a CH2 domain and a CH3 domain, and optionallycomprises a CH4 domain.

By “Fc region chain” herein is meant one of the two polypeptide chainsof an Fc region.

The “CH2 domain” of a human IgG Fc region (also referred to as “Cγ2”domain) usually extends from an amino acid residue at about position 231to an amino acid residue at about position 340. The CH2 domain is uniquein that it is not closely paired with another domain. Rather, twoN-linked branched carbohydrate chains are interposed between the two CH2domains of an intact native IgG molecule. It has been speculated thatthe carbohydrate may provide a substitute for the domain-domain pairingand help stabilize the CH2 domain. Burton, Molec. Immunol. 22:161-206(1985). The CH2 domain herein may be a native sequence CH2 domain orvariant CH2 domain.

The “CH3 domain” comprises the stretch of residues C-terminal to a CH2domain in an Fc region (i.e. from an amino acid residue at aboutposition 341 to an amino acid residue at about position 447 of an IgG).The CH3 region herein may be a native sequence CH3 domain or a variantCH3 domain (e.g. a CH3 domain with an introduced “protroberance” in onechain thereof and a corresponding introduced “cavity” in the other chainthereof; see U.S. Pat. No. 5,821,333). Such variant CH3 domains may beused to make multispecific (e.g. bispecific) antibodies as hereindescribed.

“Hinge region” is generally defined as stretching from about Glu216, orabout Cys226, to about Pro230 of human IgG1 (Burton, Molec. Immunol.22:161-206 (1985)). Hinge regions of other IgG isotypes may be alignedwith the IgG1 sequence by placing the first and last cysteine residuesforming inter-heavy chain S—S bonds in the same positions. The hingeregion herein may be a native sequence hinge region or a variant hingeregion. The two polypeptide chains of a variant hinge region generallyretain at least one cysteine residue per polypeptide chain, so that thetwo polypeptide chains of the variant hinge region can form a disulfidebond between the two chains. The preferred hinge region herein is anative sequence human hinge region, e.g. a native sequence human IgG1hinge region.

A “functional Fc region” possesses at least one “effector function” of anative sequence Fc region. Exemplary “effector functions” include C1qbinding; complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor; BCR), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g. an antibody variable domain) and can beassessed using various assays known in the art for evaluating suchantibody effector functions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of a Fc region found in nature. A “variant Fcregion” comprises an amino acid sequence which differs from that of anative sequence Fc region by virtue of at least one amino acidmodification. Preferably, the variant Fc region has at least one aminoacid substitution compared to a native sequence Fc region or to the Fcregion of a parent polypeptide, e.g. from about one to about ten aminoacid substitutions, and preferably from about one to about five aminoacid substitutions in a native sequence Fc region or in the Fc region ofthe parent polypeptide. The variant Fc region herein will preferablypossess at least about 80% sequence identity with a native sequence Fcregion and/or with an Fc region of a parent polypeptide, and mostpreferably at least about 90% sequence identity therewith, morepreferably at least about 95% sequence identity therewith.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991).To assess ADCC activity of a molecule of interest, an in vitro ADCCassay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337may be performed. Useful effector cells for such assays includeperipheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al. PNAS (USA), 95:652-656 (1998).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source thereof, e.g. from blood or PBMCs asdescribed herein.

The terms “Fc receptor” and “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. Activating receptor FcγRIIA contains animmunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmicdomain. Inhibiting receptor FcγRIIB contains an immunoreceptortyrosine-based inhibition motif (ITIM) in its cytoplasmic domain(reviewed in Daëron, Annu. Rev. Immunol., 15:203-234 (1997)). FcRs arereviewed in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991);Capel et al., Immunomethods, 4:25-34 (1994); and de Haas et al., J. Lab.Clin. Med., 126:330-41 (1995). Other FcRs, including those to beidentified in the future, are encompassed by the term “FcR” herein. Theterm also includes the neonatal receptor, FcRn, which is responsible forthe transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.,117:587 (1976); and Kim et al., J. Immunol., 24:249 (1994)).

“Complement dependent cytotoxicity” and “CDC” refer to the lysing of atarget in the presence of complement. The complement activation pathwayis initiated by the binding of the first component of the complementsystem (C1q) to a molecule (e.g. an antibody) complexed with a cognateantigen. To assess complement activation, a CDC assay, e.g. as describedin Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996), may beperformed.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result an improvement in the affinity ofthe antibody for antigen, compared to a parent antibody which does notpossess those alteration(s). Preferred affinity matured antibodies willhave nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.Marks et al. Bio/Technology, 10:779-783 (1992) describes affinitymaturation by VH and VL domain shuffling. Random mutagenesis of CDRand/or framework residues is described by: Barbas et al. Proc Nat. Acad.Sci, USA 91:3809-3813 (1994); Schier et al. Gene, 169:147-155 (1995);Yelton et al. J. Immunol., 155:1994-2004 (1995); Jackson et al., J.Immunol., 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.,226:889-896 (1992).

The term “immunospecific” as used in “immunospecific binding ofantibodies” for example, refers to the antigen specific bindinginteraction that occurs between the antigen-combining site of anantibody and the specific antigen recognized by that antibody.

The terms “cancer”, “cancerous”, “metastasis” and “malignant” refer toor describe the physiological condition in mammals that is typicallycharacterized by unregulated cell growth. Examples of cancer include butare not limited to, carcinoma including adenocarcinoma, lymphoma,blastoma, melanoma, sarcoma, and leukemia. More particular examples ofsuch cancers include squamous cell cancer, small-cell lung cancer,non-small cell lung cancer, gastrointestinal cancer, Hodgkin's andnon-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer such as hepatic carcinoma andhepatoma, bladder cancer, breast cancer, colon cancer, colorectalcancer, endometrial carcinoma, myeloma (such as multiple myeloma),salivary gland carcinoma, kidney cancer such as renal cell carcinoma andWilms' tumors, basal cell carcinoma, melanoma, prostate cancer, vulvalcancer, thyroid cancer, testicular cancer, esophageal cancer, andvarious types of head and neck cancer. The preferred cancers fortreatment herein include breast cancer, gastric cancer, lung cancer,colon or colorectal cancer, glioma and glioblastoma.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to cancer cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) andStella et al., “Prodrugs: A Chemical Approach to Targeted DrugDelivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267,Humana Press (1985). The prodrugs of this invention include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,beta-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described below.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of conditions like cancer. Examples of chemotherapeutic agentsinclude alkylating agents such as thiotepa and cyclosphosphamide(CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,ranimustine; antibiotics such as the enediyne antibiotics (e.g.calicheamicin, especially calicheamicin γ₁ ^(I) and calicheamicin θ^(I)₁, see, e.g., Agnew Chem Intl. Ed. Engl., 33:183-186 (1994); dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antibiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin,potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folicacid analogues such as denopterin, methotrexate, pteropterin,trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,doxifluridine, enocitabine, floxuridine, 5-FU; androgens such ascalusterone, dromostanolone propionate, epitiostanol, mepitiostane,testolactone; anti-adrenals such as aminoglutethimide, mitotane,trilostane; folic acid replenisher such as frolinic acid; aceglatone;aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;elformithine; elliptinium acetate; an epothilone; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such asmaytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol;nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid;2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE®, Rhône-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; capecitabine; andpharmaceutically acceptable salts, acids or derivatives of any of theabove. Also included in this definition are anti-hormonal agents thatact to regulate or inhibit hormone action on tumors such asanti-estrogens including for example tamoxifen, raloxifene, aromataseinhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and toremifene (Fareston); and anti-androgenssuch as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin;and pharmaceutically acceptable salts, acids or derivatives of any ofthe above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, either in vitro or in vivo.Thus, the growth inhibitory agent is one which significantly reduces thepercentage of cells overexpressing such genes in S phase. Examples ofgrowth inhibitory agents include agents that block cell cycleprogression (at a place other than S phase), such as agents that induceG1 arrest and M-phase arrest. Classical M-phase blockers include thevincas (vincristine and vinblastine), taxol, and topo II inhibitors suchas doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest G1 also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogens, and antineoplastic drugs” by Murakami et al. (WBSaunders: Philadelphia, 1995), especially p. 13.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;Apo-2 ligand (also referred to as TRAIL); mullerian-inhibitingsubstance; mouse gonadotropin-associated peptide; inhibin; activin;vascular endothelial growth factor; integrin; thrombopoietin (TPO);nerve growth factors; platelet-growth factor; transforming growthfactors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and-II; erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -gamma; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; and other polypeptidefactors including LIF and kit ligand (KL). As used herein, the termcytokine includes proteins from natural sources or from recombinant cellculture and biologically active equivalents of the native sequencecytokines.

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology of a disorder.Accordingly, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) the targeted pathological condition or disorder.Those in need of treatment include those already with the disorder aswell as those in which the disorder is to be prevented. In treatment ofa disorder, a therapeutic agent may directly decrease or increase themagnitude of response of a pathological component of the disorder, orrender the disease more susceptible to treatment by other therapeuticagents, e.g. antibiotics, antifungals, anti-inflammatory agents,chemotherapeutics, etc.

The term “effective amount” is the minimum concentration of WISP-1antagonist which causes, induces or results in either a detectableimprovement or reduction of a pathological condition. In a method oftreating cancer, an effective amount is one which causes, induces, orresults in reduction of cancer cell number or tumor volume. Furthermorea “therapeutically effective amount” is the minimum concentration(amount) of WISP-1 antagonist administered to a mammal which would beeffective in at least attenuating a pathological symptom.

“Chronic” administration refers to administration of the factor(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is done notconsecutively without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cattle, pigs, hamsters, etc.Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN®, polyethylene glycol (PEG), and PLURONICS®, hyaluronicacid (HA).

II. Methods and Compositions of the Invention

As described further in the Examples below, Applicant has surprisinglyfound that ectopic expression of WISP-1 in fibroblasts triggered HAproduction, namely increased HA production induced its accumulation atthe cell surface and in the culture media. It is believed that cellsurface HA can enhance anchorage-independent growth and tumorigenicity(Kosaki et al., Cancer Res., 59:1141-1145 (1999)) and may diminishcontact inhibition of cell proliferation, thereby promoting cellmigration (Ichikawa et al., J. Invest. Dermatol., 113:935-939 (1999);Itano et al., Proc. Natl. Acad. Sci., 99:3609-3614 (2002)). Hyaluronancoat formation has also been correlated with cellular metastaticpotential (Zhang et al., Cancer Res., 55:428-433 (1995); Toole et al.,J. Biol. Chem., 277:4593-4596 (2002)). Analysis of HA synthases (HAS1,HAS2, and HAS3) expression revealed that WISP-1 promoted HAS2 inductionwhereas HAS1 and HAS3 mRNA levels remained unchanged. HAS2 is the maintarget of growth factor and cytokine up-regulated HA synthesis(Pienimaki et al., J. Biol. Chem., 276:20428-20435 (2001)). Ectopicexpression of HAS2 increases HA secretion and induces hyaluronan coatformation (Kosaki et al., supra). Moreover, HAS2 is believed to beimportant for hyaluronan-mediated transformation of epithelium tomesenchyme during cardiac morphogenesis (Camenisch et al., J. Clin.Invest., 106:349-360 (2000)). Because epithelial to mesenchymaltransition is also important for tumor invasion and metastasis, HAS2 ispostulated to play an important role in epithelial tumor progression(Boyer et al., Biochem. Pharmacol., 60:1091-1099 (2000); Hay, ActaAnat., 154:8-20 (1995); Arias, Cell, 105:425-431 (2001)). These resultsindicate that HA secretion induced by WISP-1 may be important for tumorinvasion and metastasis.

WISP-1 also induced the expression of two HA receptors, CD44 and RHAMM.By inducing HA receptor expression and increasing HA production, WISP-1may activate an autocrine and/or paracrine loop. Hyaluronan interactionwith CD44 and RHAMM promotes cell locomotion and proliferation in vitroand tumor growth and metastasis in vivo (Turley et al., J. Biol. Chem.,277:4589-4592 (2002); Sy et al., Curr. Top. Microbiol. Immunol.,213:129-153 (1996); Hall et al., J. Neurooncol., 26:221-229 (1995)).

The effects of WISP-1 on cell migration were analyzed in a cell woundhealing assay and by time lapse microscopy, and the data showed thatWISP-1 expression promoted cell motility. Further, isolated cells showedincreased migration suggesting that WISP-1 could act through anautocrine mechanism. Purified recombinant WISP-1 promoted HAS2 and CD44expression, though, when attached to a surface. This induction wasfurther increased when WISP-1 was tethered through its interaction withdecorin. Similarly, WISP-1 promoted cell motility when surface bound,suggesting that WISP-1 may also act through a paracrine mechanism whentethered to a substrate. The Examples herein show that WISP-1 inducedcell motility was mediated by CD44, by abolishing migration with ananti-CD44 antibody. WISP-1 induced cell motility was also inhibited byWISP-1 antibodies. Moreover, WISP-1 haptotactic activity was notrestricted to a single cell type as it induced both normal fibroblastand colon adenocarcinoma cell migration.

Because WISP-1 is believed to be a Wnt-1 downstream effector, theC57MG/Wnt-1 cell line was analyzed for phenotypes found in NRK/WISP-1cells. Consistent with a role for WISP-1 downstream of Wnt-1,C57MG/Wnt-1 cells overexpressed HAS2 and CD44 and had higher CD44protein content compared to the parental cell line. In addition,C57/Wnt-1 cells spontaneously scattered in culture and demonstrated ade-differentiated spindloid morphology similar to NRK/WISP-1H cells.

Expression analysis was also performed on a group of MMTV-Wnt-1 mammarytumors, and elevated CD44 and HAS2 expression was detected in allmammary tumors from the MMTV-Wnt-1 transgenic mice. In these tumors,WISP-1 expression was localized to the stromal fibroblasts whereas CD44and HAS2 were expressed by tumor epithelial cells. Although negative forHAS2 expression, the peritumoral stroma contained high levels of HAwhereas the tumor parenchyma stained only weakly for HA. Although it isgenerally accepted that fibroblasts are responsible for HA production,the origin of stromal HA is not definitely known. Because HAS2expression was found only in the parenchyma, the experimental resultsdisclosed herein suggest that the tumor cells are responsible for the HAsynthesis and deposition in the stroma. Hyaluronan accumulation in theperitumoral stroma is frequently encountered in several tumor types andwas previously reported for ovary, breast, prostate and colonadenocarcinomas (Ropponen et al., Cancer Res., 58:342-347 (1998);Lipponen et al., Eur. J. Cancer, 37:849-856 (2001); Auvinen et al., Am.J. Pathol., 156:529-536 (2000); Anttila et al., Cancer Res., 60:150-155(2000)). Moreover, a high stromal level of HA was associated with poordifferentiation, metastatic behavior and unfavorable prognosis.

Because HA and CD44 are associated with tumor invasion, the metastaticpotential of WISP-1 expressing cells was evaluated in vivo. After tailinoculation, NRK/WISP-1 cells readily colonized the lungs of injectedmice and formed invasive tumors. NRK/WISP-1 cells exhibited asignificant metastatic potential while NRK cells did not. Histologicalobservation revealed that NRK/WISP-1 cells populated the vasculature andinvaded the pulmonary airways. The lung colonization was proportional tothe number of cells injected, the time after injection and the WISP-1expression. In addition, Applicant found the metastatic potential to beproportional to CD44 and HAS2 expression levels, and the treatment ofmice inoculated with NRK/WISP-1H cells with CD44 antibody or WISP-1antibody greatly diminished the number and size of the tumors in thelung. Thus, it is likely that levels of WISP-1 promoted lungcolonization through a hyaluronan-CD44 mechanism. This is consistentwith previous reports demonstrating the importance of CD44 and itsinteraction with HA for tumor growth, metastasis and the retention ofmetastatic cells to the lung vasculature (Sy et al., supra; Kogerman etal., Proc. Natl. Acad. Sci., 94:13233-13238 (1997)).

Although activation of the Wnt pathway by APC or β-catenin mutations maytypically be associated with colorectal cancer, several lines ofevidence suggest that it also plays a role in other types of cancerincluding mammary adenocarcinoma (Polakis, Genes Dev., 14:1837-1851(2000); Brown, Breast Cancer Res., 3:351-355 (2001)). APC truncation andincreased β-catenin levels were found in human breast cancer cell lines(Schlosshauer et al., Carcinogenesis, 21:1453-1456 (2000)). Somaticmutations of the APC gene were found in primary breast cancers (Furuuchiet al., Am. J. Pathol., 156:1997-2005 (2000)). In addition, β-cateninactivating mutations promote mouse mammary adenocarcinomas Michaelson etal., Oncogene, 20:5525-5532 (2001)). Elevated levels of Wnt-1 andβ-catenin were found in invasive ductal breast carcinomas and correlatedwith poor prognosis (Lin et al., Proc. Natl. Acad. Sci., 97:4262-4266(2000)). It is therefore possible that WISP-1 expression found incertain breast adenocarcinomas resulted from Wnt pathway activation.Applicants have also found, by Taqman analysis, that WISP-1 isoverexpressed in cancers such as breast and glial tumors.

For at least these reasons, it is believed that WISP-1 antgonists willbe particularly useful in treating and diagnosing various pathologicaldisorders, such as cancer. The present invention accordingly providesmethods for modulating, blocking or neutralizing WISP-1 activity inmammalian cells which comprise exposing the cells to a desired amount ofWISP-1 antagonist. Preferably, the amount of WISP-1 antagonist employedwill be an amount effective to reduce or inhibit cancer cell growth,metastasis or motility. This can be accomplished in vivo or ex vivo inaccordance, for instance, with the methods described below and in theExamples. Exemplary conditions or disorders to be treated with suchWISP-1 antagonists include conditions in mammals clinically referred toas cancer.

Diagnostic methods are also provided herein. For instance, theantagonists may be employed to detect invasive or metastatic cancers.The antagonist molecule may be used, e.g., in assays to detect orquantitate metastatic cancer cells in a sample. A sample, such as cellsobtained from a mammal, can be incubated in the presence of a labeledantagonist, and detection of the labeled antagonist bound in the samplecan be performed.

The antagonists which can be employed in the methods include, but arenot limited to, WISP-1 immunoadhesins, fusion proteins comprisingWISP-1, covalently modified forms of WISP-1, WISP-1 variants, fusionproteins thereof, and WISP-1 antibodies. Various techniques that can beemployed for making the antagonists are described herein. For instance,methods and techniques for preparing WISP-1 polypeptides are described.Further modifications of the polypeptides, and antibodies to WISP-1 arealso described.

In addition to the full-length native sequence WISP-1 polypeptidedescribed herein, it is contemplated that WISP-1 polypeptide variantscan be prepared. WISP-1 variants can be prepared by introducingappropriate nucleotide changes into the encoding DNA, and/or bysynthesis of the desired polypeptide. Those skilled in the art willappreciate that amino acid changes may alter post-translationalprocesses of the WISP-1 polypeptide, such as changing the number orposition of glycosylation sites or altering the membrane anchoringcharacteristics.

Variations in the WISP-1 polypeptides described herein, can be made, forexample, using any of the techniques and guidelines for conservative andnon-conservative mutations set forth, for instance, in U.S. Pat. No.5,364,934. Variations may be a substitution, deletion or insertion ofone or more codons encoding the polypeptide that results in a change inthe amino acid sequence as compared with the native sequencepolypeptide. Optionally the variation is by substitution of at least oneamino acid with any other amino acid in one or more of the domains ofthe WISP-1 polypeptide. Guidance in determining which amino acid residuemay be inserted, substituted or deleted without adversely affecting thedesired activity may be found by comparing the sequence of the WISP-1polypeptide with that of homologous known protein molecules andminimizing the number of amino acid sequence changes made in regions ofhigh homology. Amino acid substitutions can be the result of replacingone amino acid with another amino acid having similar structural and/orchemical properties, such as the replacement of a leucine with a serine,i.e., conservative amino acid replacements. Insertions or deletions mayoptionally be in the range of about 1 to 5 amino acids. The variationallowed may be determined by systematically making insertions, deletionsor substitutions of amino acids in the sequence and testing theresulting variants for activity exhibited by the full-length or maturenative sequence.

WISP-1 polypeptide fragments are provided herein. Such fragments may betruncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native protein.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the WISP-1 polypeptide.

WISP-1 polypeptide fragments may be prepared by any of a number ofconventional techniques. Desired peptide fragments may be chemicallysynthesized. An alternative approach involves generating polypeptidefragments by enzymatic digestion, e.g., by treating the protein with anenzyme known to cleave proteins at sites defined by particular aminoacid residues, or by digesting the DNA with suitable restriction enzymesand isolating the desired fragment. Yet another suitable techniqueinvolves isolating and amplifying a DNA fragment encoding a desiredpolypeptide fragment, by polymerase chain reaction (PCR).Oligonucleotides that define the desired termini of the DNA fragment areemployed at the 5′ and 3′ primers in the PCR.

In particular embodiments, conservative substitutions of interest areshown in the Table below under the heading of preferred substitutions.If such substitutions result in a change in biological activity, thenmore substantial changes, denominated exemplary substitutions in theTable, or as further described below in reference to amino acid classes,are introduced and the products screened. TABLE Original ExemplaryPreferred Residue Substitutions Substitutions Ala (A) val; leu; ile valArg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu gluCys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His(H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; leunorleucine Leu (L) norleucine; ile; val; ile met; ala; phe Lys (K) arg;gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyrleu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyrTyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; leu ala;norleucine

Substantial modifications in function or immunological identity of theWISP-1 polypeptide are accomplished by selecting substitutions thatdiffer significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of the substitution, for example,as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.Naturally occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the WISP-1 polypeptide variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244:1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

Any cysteine residue not involved in maintaining the proper conformationof the WISP-1 polypeptide also may be substituted, generally withserine, to improve the oxidative stability of the molecule and preventaberrant crosslinking. Conversely, cysteine bond(s) may be added to theWISP-1 polypeptide to improve its stability.

The description below relates primarily to production of WISP-1polypeptides by culturing cells transformed or transfected with a vectorcontaining WISP-1 polypeptide-encoding nucleic acid. It is, of course,contemplated that alternative methods, which are well known in the art,may be employed to prepare WISP-1 polypeptides. For instance, theappropriate amino acid sequence, or portions thereof, may be produced bydirect peptide synthesis using solid-phase techniques [see, e.g.,Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., SanFrancisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154(1963)]. In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be accomplished,for instance, using an Applied Biosystems Peptide Synthesizer (FosterCity, Calif.) using manufacturer's instructions. Various portions of theWISP-1 polypeptide may be chemically synthesized separately and combinedusing chemical or enzymatic methods to produce the desired WISP-1polypeptide. The methods and techniques described are similarlyapplicable to production of WISP-1 variants, modified forms of WISP-1and WISP-1 antibodies.

1. Isolation of DNA Encoding WISP-1 Polypeptide

DNA encoding WISP-1 polypeptide may be obtained from a cDNA libraryprepared from tissue believed to possess the WISP-1 polypeptide mRNA andto express it at a detectable level. Accordingly, human WISP-1polypeptide DNA can be conveniently obtained from a cDNA libraryprepared from human tissue. The WISP-1 polypeptide-encoding gene mayalso be obtained from a genomic library or by known synthetic procedures(e.g., automated nucleic acid synthesis).

Libraries can be screened with probes (such as oligonucleotides of atleast about 20-80 bases) designed to identify the gene of interest orthe protein encoded by it. Screening the cDNA or genomic library withthe selected probe may be conducted using standard procedures, such asdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual(New York: Cold Spring Harbor Laboratory Press, 1989). An alternativemeans to isolate the gene encoding WISP-1 polypeptide is to use PCRmethodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

Techniques for screening a cDNA library are well known in the art. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined using methods known in the art and as described herein.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for WISP-1 polypeptide production and culturedin conventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7ilvG kan^(r) ; E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued 7 Aug. 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for WISP-1polypeptide-encoding vectors. Saccharomyces cerevisiae is a commonlyused lower eukaryotic host microorganism. Others includeSchizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Pat. No.4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such as,e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J.Bacteriol., 154(2):737-742 [1983]), K. fragilis (ATCC 12,424), K.bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al.,Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus;yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al.,J. Basic Microbiol., 28:265-278 [1988]); Candida; Trichoderma reesia (EP244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA,76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis(EP 394,538 published 31 Oct. 1990); and filamentous fungi such as,e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10Jan. 1991), and Aspergillus hosts such as A. nidulans (Ballance et al.,Biochem. Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al.,Gene, 26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated WISP-1polypeptide are derived from multicellular organisms. Examples ofinvertebrate cells include insect cells such as Drosophila S2 andSpodoptera Sf9, as well as plant cells, such as cell cultures of cotton,corn, potato, soybean, petunia, tomato, and tobacco. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for WISP-1 polypeptide production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding WISP-1 polypeptidemay be inserted into a replicable vector for cloning (amplification ofthe DNA) or for expression. Various vectors are publicly available. Thevector may, for example, be in the form of a plasmid, cosmid, viralparticle, or phage. The appropriate nucleic acid sequence may beinserted into the vector by a variety of procedures. In general, DNA isinserted into an appropriate restriction endonuclease site(s) usingtechniques known in the art. Vector components generally include, butare not limited to, one or more of a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence. Construction of suitablevectors containing one or more of these components employs standardligation techniques which are known to the skilled artisan.

The WISP-1 may be produced recombinantly not only directly, but also asa fusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe WISP-1 polypeptide-encoding DNA that is inserted into the vector.The signal sequence may be a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, lpp,or heat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), orthe signal described in WO 90/13646 published 15 Nov. 1990. In mammaliancell expression, mammalian signal sequences may be used to directsecretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2 μl plasmid origin is suitable for yeast,and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) areuseful for cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up the WISP-1polypeptide-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the WISP-1 polypeptide-encoding nucleic acid sequence todirect mRNA synthesis. Promoters recognized by a variety of potentialhost cells are well known. Promoters suitable for use with prokaryotichosts include the β-lactamase and lactose promoter systems [Chang etal., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)],alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel,Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters suchas the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25(1983)]. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding WISPpolypeptide.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

WISP polypeptide transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus,avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virusand Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g.,the actin promoter or an immunoglobulin promoter, and from heat-shockpromoters, provided such promoters are compatible with the host cellsystems.

Transcription of a DNA encoding the WISP-1 polypeptide by highereukaryotes may be increased by inserting an enhancer sequence into thevector. Enhancers are cis-acting elements of DNA, usually about from 10to 300 bp, that act on a promoter to increase its transcription. Manyenhancer sequences are now known from mammalian genes (globin, elastase,albumin, -fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theWISP-1 polypeptide coding sequence, but is preferably located at a site5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding WISP-1 polypeptide.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of WISP polypeptide in recombinant vertebrate cell cultureare described in Gething et al., Nature, 293:620-625 (1981); Mantei etal., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

4. Culturing the Host Cells

The host cells used to produce the WISP polypeptide of this inventionmay be cultured in a variety of media. Commercially available media suchas Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma),RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM),Sigma) are suitable for culturing the host cells. In addition, any ofthe media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes etal., Anal. Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866;4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S.Pat. Re. 30,985 may be used as culture media for the host cells. Any ofthese media may be supplemented as necessary with hormones and/or othergrowth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

5. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequenceWISP polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to WISPDNA and encoding a specific antibody epitope.

6. Purification of WISP Polypeptide

Forms of WISP polypeptide may be recovered from culture medium or fromhost cell lysates. If membrane-bound, it can be released from themembrane using a suitable detergent solution (e.g. Triton-X 100) or byenzymatic cleavage. Cells employed in expression of WISP-1 polypeptidecan be disrupted by various physical or chemical means, such asfreeze-thaw cycling, sonication, mechanical disruption, or cell lysingagents.

It may be desired to purify WISP-1 polypeptide from recombinant cellproteins or polypeptides. The following procedures are exemplary ofsuitable purification procedures: by fractionation on an ion-exchangecolumn; ethanol precipitation; reverse phase HPLC; chromatography onsilica or on a cation-exchange resin such as DEAE; chromatofocusing;SDS-PAGE; ammonium sulfate precipitation; gel filtration using, forexample, Sephadex G-75; protein A Sepharose columns to removecontaminants such as IgG; and metal chelating columns to bindepitope-tagged forms of the WISP-1 polypeptide. Various methods ofprotein purification may be employed and such methods are known in theart and described for example in Deutscher, Methods in Enzymology, 182(1990); Scopes, Protein Purification: Principles and Practice,Springer-Verlag, New York (1982). The purification step(s) selected willdepend, for example, on the nature of the production process used andthe particular WISP-1 polypeptide produced.

Soluble forms of WISP-1 may be employed as antagonists in the methods ofthe invention. Such soluble forms of WISP-1 may comprise modifications,as described below (such as by fusing to an immunoglobulin, epitope tagor leucine zipper). Immunoadhesin molecules are further contemplated foruse in the methods herein. WISP-1 immunoadhesins may comprise variousforms of WISP-1, such as the full length polypeptide as well as solubleforms of the WISP-1 or a fragment thereof. In particular embodiments,the molecule may comprise a fusion of the WISP-1 polypeptide with animmunoglobulin or a particular region of an immunoglobulin. For abivalent form of the immunoadhesin, such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of the polypeptide in place of at least one variable region withinan Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions, see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995 and Chamow et al., TIBTECH, 14:52-60 (1996).

The simplest and most straightforward immunoadhesin design combines thebinding domain(s) of the adhesin (e.g. the WISP-1) with the Fc region ofan immunoglobulin heavy chain. Ordinarily, when preparing theimmunoadhesins of the present invention, nucleic acid encoding thebinding domain of the adhesin will be fused C-terminally to nucleic acidencoding the N-terminus of an immunoglobulin constant domain sequence,however N-terminal fusions are also possible.

Typically, in such fusions the encoded chimeric polypeptide will retainat least functionally active hinge, C_(H)2 and C_(H)3 domains of theconstant region of an immunoglobulin heavy chain. Fusions are also madeto the C-terminus of the Fc portion of a constant domain, or immediatelyN-terminal to the C_(H)1 of the heavy chain or the corresponding regionof the light chain. The precise site at which the fusion is made is notcritical; particular sites are well known and may be selected in orderto optimize the biological activity, secretion, or bindingcharacteristics of the immunoadhesin.

In a preferred embodiment, the adhesin sequence is fused to theN-terminus of the Fc region of immunoglobulin G₁ (IgG₁). It is possibleto fuse the entire heavy chain constant region to the adhesin sequence.However, more preferably, a sequence beginning in the hinge region justupstream of the papain cleavage site which defines IgG Fc chemically(i.e. residue 216, taking the first residue of heavy chain constantregion to be 114), or analogous sites of other immunoglobulins is usedin the fusion. In a particularly preferred embodiment, the adhesin aminoacid sequence is fused to (a) the hinge region and C_(H)2 and C_(H)3 or(b) the C_(H)1, hinge, C_(H)2 and C_(H)3 domains, of an IgG heavy chain.

For bispecific immunoadhesins, the immunoadhesins are assembled asmultimers, and particularly as heterodimers or heterotetramers.Generally, these assembled immunoglobulins will have known unitstructures. A basic four chain structural unit is the form in which IgG,IgD, and IgE exist. A four chain unit is repeated in the highermolecular weight immunoglobulins; IgM generally exists as a pentamer offour basic units held together by disulfide bonds. IgA globulin, andoccasionally IgG globulin, may also exist in multimeric form in serum.In the case of multimer, each of the four units may be the same ordifferent.

Various exemplary assembled immunoadhesins within the scope herein areschematically diagrammed below:

(a) AC_(L)-AC_(L);

(b) AC_(H)-(AC_(H), AC_(L)-AC_(H), AC_(L)-V_(H)C_(H), orV_(L)C_(L)-AC_(H));

(c) AC_(L)-AC_(H)-(AC_(L)-AC_(H), AC_(L)-V_(H)C_(H), V_(L)C_(L)-AC_(H),or V_(L)C_(L)-V_(H)C_(H))

(d) AC_(L)-V_(H)C_(H)-(AC_(H), or AC_(L)-V_(H)C_(H), orV_(L)C_(L)-AC_(H));

(e) V_(L)C_(L)-AC_(H)-(AC_(L)-V_(H)C_(H), or V_(L)C_(L)-AC_(H)); and

(f) (A-Y)_(n)-(V_(L)C_(L)-V_(H)C_(H))₂,

wherein each A represents identical or different adhesin amino acidsequences;

V_(L) is an immunoglobulin light chain variable domain;

V_(H) is an immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H) is an immunoglobulin heavy chain constant domain;

n is an integer greater than 1;

Y designates the residue of a covalent cross-linking agent.

In the interests of brevity, the foregoing structures only show keyfeatures; they do not indicate joining (J) or other domains of theimmunoglobulins, nor are disulfide bonds shown. However, where suchdomains are required for binding activity, they shall be constructed tobe present in the ordinary locations which they occupy in theimmunoglobulin molecules.

Alternatively, the adhesin sequences can be inserted betweenimmunoglobulin heavy chain and light chain sequences, such that animmunoglobulin comprising a chimeric heavy chain is obtained. In thisembodiment, the adhesin sequences are fused to the 3′ end of animmunoglobulin heavy chain in each arm of an immunoglobulin, eitherbetween the hinge and the C_(H)2 domain, or between the C_(H)2 andC_(H)3 domains. Similar constructs have been reported by Hoogenboom etal., Mol. Immunol., 28:1027-1037 (1991).

Although the presence of an immunoglobulin light chain is not requiredin the immunoadhesins of the present invention, an immunoglobulin lightchain might be present either covalently associated to anadhesin-immunoglobulin heavy chain fusion polypeptide, or directly fusedto the adhesin. In the former case, DNA encoding an immunoglobulin lightchain is typically coexpressed with the DNA encoding theadhesin-immunoglobulin heavy chain fusion protein. Upon secretion, thehybrid heavy chain and the light chain will be covalently associated toprovide an immunoglobulin-like structure comprising two disulfide-linkedimmunoglobulin heavy chain-light chain pairs. Methods suitable for thepreparation of such structures are, for example, disclosed in U.S. Pat.No. 4,816,567, issued 28 Mar. 1989.

Immunoadhesins are most conveniently constructed by fusing the cDNAsequence encoding the adhesin portion in-frame to an immunoglobulin cDNAsequence. However, fusion to genomic immunoglobulin fragments can alsobe used (see, e.g. Aruffo et al., Cell, 61:1303-1313 (1990); andStamenkovic et al., Cell, 66:1133-1144 (1991)). The latter type offusion requires the presence of Ig regulatory sequences for expression.cDNAs encoding IgG heavy-chain constant regions can be isolated based onpublished sequences from cDNA libraries derived from spleen orperipheral blood lymphocytes, by hybridization or by polymerase chainreaction (PCR) techniques. The cDNAs encoding the “adhesin” and theimmunoglobulin parts of the immunoadhesin are inserted in tandem into aplasmid vector that directs efficient expression in the chosen hostcells.

In another embodiment, the WISP-1 or WISP-1 antagonist may be covalentlymodified by linking the receptor polypeptide to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337, or other like molecules such as polyglutamate. Such pegylatedforms may be prepared using techniques known in the art.

Leucine zipper forms of these molecules are also contemplated by theinvention. “Leucine zipper” is a term in the art used to refer to aleucine rich sequence that enhances, promotes, or drives dimerization ortrimerization of its fusion partner (e.g., the sequence or molecule towhich the leucine zipper is fused or linked to). Various leucine zipperpolypeptides have been described in the art. See, e.g., Landschulz etal., Science, 240:1759 (1988); U.S. Pat. No. 5,716,805; WO 94/10308;Hoppe et al., FEBS Letters, 344:1991 (1994); Maniatis et al., Nature,341:24 (1989). Those skilled in the art will appreciate that a leucinezipper sequence may be fused at either the 5′ or 3′ end of the WISP-1 orWISP-1 antagonist molecule.

The WISP-1 polypeptides of the present invention may also be modified ina way to form chimeric molecules by fusing the polypeptide to another,heterologous polypeptide or amino acid sequence. Preferably, suchheterologous polypeptide or amino acid sequence is one which acts tooligimerize the chimeric molecule. In one embodiment, such a chimericmolecule comprises a fusion of the WISP-1 polypeptide with a tagpolypeptide which provides an epitope to which an anti-tag antibody canselectively bind. The epitope tag is generally placed at the amino- orcarboxyl-terminus of the polypeptide. The presence of suchepitope-tagged forms of the polypeptide can be detected using anantibody against the tag polypeptide. Also, provision of the epitope tagenables the polypeptide to be readily purified by affinity purificationusing an anti-tag antibody or another type of affinity matrix that bindsto the epitope tag. Various tag polypeptides and their respectiveantibodies are well known in the art. Examples include poly-histidine(poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tagpolypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol.,8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an -tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

It is contemplated that anti-WISP-1 antibodies may also be employed inthe presently disclosed methods. Examples of such molecules includeneutralizing or blocking antibodies which can reduce cancer cell growth,metastasis or motility. The anti-WISP-1 may be monoclonal antibodies.

Monoclonal antibodies may be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes may beimmunized in vitro.

The immunizing agent will typically include a WISP-1 polypeptide or afusion protein thereof, such as a WISP-1-IgG fusion protein. Generally,either peripheral blood lymphocytes (“PBLs”) are used if cells of humanorigin are desired, or spleen cells or lymph node cells are used ifnon-human mammalian sources are desired. The lymphocytes are then fusedwith an immortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells may becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed againstWISP-1. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods[Goding, supra]. Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the monoclonal antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. The DNA also may be modified, for example, by substituting thecoding sequence for human heavy and light chain constant domains inplace of the homologous murine sequences, Morrison, et al., Proc. Nat.Acad. Sci. 81, 6851 (1984), or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for WISP-1 andanother antigen-combining site having specificity for a differentantigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

Single chain Fv fragments may also be produced, such as described inIliades et al., FEBS Letters, 409:437-441 (1997). Coupling of suchsingle chain fragments using various linkers is described in Kortt etal., Protein Engineering, 10:423-433 (1997). A variety of techniques forthe recombinant production and manipulation of antibodies are well knownin the art. Illustrative examples of such techniques that are typicallyutilized by skilled artisans are described in greater detail below.

(i) Humanized Antibodies

Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a non-human source. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers [Jones et al.,Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody.

Accordingly, such “humanized” antibodies are chimeric antibodies whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

It is important that antibodies be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, according to a preferred method, humanized antibodiesare prepared by a process of analysis of the parental sequences andvarious conceptual humanized products using three dimensional models ofthe parental and humanized sequences. Three dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e. the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from theconsensus and import sequence so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding.

(ii) Human Antibodies

Human monoclonal antibodies can be made by the hybridoma method. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described, for example, by Kozbor,J. Immunol. 133, 3001 (1984), and Brodeur, et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g. Jakobovits et al.,Proc. Natl. Acad. Sci. USA 90, 2551-255 (1993); Jakobovits et al.,Nature 362, 255-258 (1993).

Mendez et al. (Nature Genetics 15: 146-156 [1997]) have further improvedthe technology and have generated a line of transgenic mice designatedas “Xenomouse II” that, when challenged with an antigen, generates highaffinity fully human antibodies. This was achieved by germ-lineintegration of megabase human heavy chain and light chain loci into micewith deletion into endogenous J_(H) segment as described above. TheXenomouse II harbors 1,020 kb of human heavy chain locus containingapproximately 66 V_(H) genes, complete D_(H) and J_(H) regions and threedifferent constant regions (μ, δ and χ), and also harbors 800 kb ofhuman κ locus containing 32 Vκ genes, Jκ segments and Cκ genes. Theantibodies produced in these mice closely resemble that seen in humansin all respects, including gene rearrangement, assembly, and repertoire.The human antibodies are preferentially expressed over endogenousantibodies due to deletion in endogenous J_(H) segment that preventsgene rearrangement in the murine locus.

Alternatively, the phage display technology (McCafferty et al., Nature348, 552-553 [1990]) can be used to produce human antibodies andantibody fragments in vitro, from immunoglobulin variable (V) domaingene repertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimicks someof the properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g. Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology 3, 564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature 352, 624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222, 581-597 (1991), or Griffith et al., EMBO J.12, 725-734 (1993). In a natural immune response, antibody genesaccumulate mutations at a high rate (somatic hypermutation). Some of thechanges introduced will confer higher affinity, and B cells displayinghigh-affinity surface immunoglobulin are preferentially replicated anddifferentiated during subsequent antigen challenge. This natural processcan be mimicked by employing the technique known as “chain shuffling”(Marks et al., Bio/Technol. 10, 779-783 [1992]). In this method, theaffinity of “primary” human antibodies obtained by phage display can beimproved by sequentially replacing the heavy and light chain V regiongenes with repertoires of naturally occurring variants (repertoires) ofV domain genes obtained from unimmunized donors. This technique allowsthe production of antibodies and antibody fragments with affinities inthe nM range. A strategy for making very large phage antibodyrepertoires (also known as “the mother-of-all libraries”) has beendescribed by Waterhouse et al., Nucl. Acids Res. 21, 2265-2266 (1993).Gene shuffling can also be used to derive human antibodies from rodentantibodies, where the human antibody has similar affinities andspecificities to the starting rodent antibody. According to this method,which is also referred to as “epitope imprinting”, the heavy or lightchain V domain gene of rodent antibodies obtained by phage displaytechnique is replaced with a repertoire of human V domain genes,creating rodent-human chimeras. Selection on antigen results inisolation of human variable capable of restoring a functionalantigen-binding site, i.e. the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT patentapplication WO 93/06213, published 1 Apr. 1993). Unlike traditionalhumanization of rodent antibodies by CDR grafting, this techniqueprovides completely human antibodies, which have no framework or CDRresidues of rodent origin.

As discussed below, the antibodies of the invention may optionallycomprise monomeric, antibodies, dimeric antibodies, as well asmultivalent forms of antibodies. Those skilled in the art may constructsuch dimers or multivalent forms by techniques known in the art. Methodsfor preparing monovalent antibodies are also well known in the art. Forexample, one method involves recombinant expression of immunoglobulinlight chain and modified heavy chain. The heavy chain is truncatedgenerally at any point in the Fc region so as to prevent heavy chaincrosslinking. Alternatively, the relevant cysteine residues aresubstituted with another amino acid residue or are deleted so as toprevent crosslinking.

(iii) Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forWISP-1. For example, bispecific antibodies specifically binding WISP-1or WISP-1 variants and another CNN family member (e.g., WISP-2, WISP-3,CTGF, Cyr61, or Nov) or other molecules such as CD44 are within thescope of the present invention.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the coexpression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities(Millstein and Cuello, Nature 305, 537-539 (1983)). Because of therandom assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of 10 differentantibody molecules, of which only one has the correct bispecificstructure. The purification of the correct molecule, which is usuallydone by affinity chromatography steps, is rather cumbersome, and theproduct yields are low. Similar procedures are disclosed in PCTapplication publication No. WO 93/08829 (published 13 May 1993), and inTraunecker et al., EMBO 10, 3655-3659 (1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH2 and CH3 regions. Itis preferred to have the first heavy chain constant region (CH1)containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are cotransfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance. In a preferred embodiment of this approach,the bispecific antibodies are composed of a hybrid immunoglobulin heavychain with a first binding specificity in one arm, and a hybridimmunoglobulin heavy chain-light chain pair (providing a second bindingspecificity) in the other arm. It was found that this asymmetricstructure facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations, as the presence of animmunoglobulin light chain in only one half of the bispecific moleculeprovides for a facile way of separation. This approach is disclosed inPCT Publication No. WO 94/04690, published on Mar. 3, 1994.

For further details of generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology 121, 210 (1986).

(iv) Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (PCT application publication Nos. WO91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

(v) Antibody Fragments

In certain embodiments, the anti-WISP-1 antibody (including murine,human and humanized antibodies, and antibody variants) is an antibodyfragment. Various techniques have been developed for the production ofantibody fragments. Traditionally, these fragments were derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al.,Science 229:81 (1985)). However, these fragments can now be produceddirectly by recombinant host cells. For example, Fab′-SH fragments canbe directly recovered from E. coli and chemically coupled to formF(ab′)₂ fragments (Carter et al., Bio/Technology 10:163-167 (1992)). Inanother embodiment, the F(ab′)₂ is formed using the leucine zipper GCN4to promote assembly of the F(ab′)₂ molecule. According to anotherapproach, Fv, Fab or F(ab′)₂ fragments can be isolated directly fromrecombinant host cell culture. A variety of techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. For instance, digestion can be performed using papain.Examples of papain digestion are described in WO 94/29348 published Dec.22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion of antibodiestypically produces two identical antigen binding fragments, called Fabfragments, each with a single antigen binding site, and a residual Fcfragment. Pepsin treatment yields an F(ab′)₂ fragment that has twoantigen combining sites and is still capable of cross-linking antigen.

The Fab fragments produced in the antibody digestion also contain theconstant domains of the light chain and the first constant domain (CH₁)of the heavy chain. Fab′ fragments differ from Fab fragments by theaddition of a few residues at the carboxy terminus of the heavy chainCH₁ domain including one or more cysteines from the antibody hingeregion. Fab′-SH is the designation herein for Fab′ in which the cysteineresidue(s) of the constant domains bear a free thiol group. F(ab′)₂antibody fragments originally were produced as pairs of Fab′ fragmentswhich have hinge cysteines between them. Other chemical couplings ofantibody fragments are also known.

Antibodies are glycosylated at conserved positions in their constantregions (Jefferis and Lund, Chem. Immunol. 65:111-128 [1997]; Wright andMorrison, TibTECH 15:26-32 [1997]). The oligosaccharide side chains ofthe immunoglobulins affect the protein's function (Boyd et al., Mol.Immunol. 32:1311-1318 [1996]; Wittwe and Howard, Biochem. 29:4175-4180[1990]), and the intramolecular interaction between portions of theglycoprotein which can affect the conformation and presentedthree-dimensional surface of the glycoprotein (Hefferis and Lund, supra;Wyss and Wagner, Current Opin. Biotech. 7:409-416 [1996)).Oligosaccharides may also serve to target a given glycoprotein tocertain molecules based upon specific recognition structures. Forexample, it has been reported that in agalactosylated IgG, theoligosaccharide moiety ‘flips’ out of the inter-CH2 space and terminalN-acetylglucosamine residues become available to bind mannose bindingprotein (Malhotra et al., Nature Med. 1:237-243 [1995)). Removal byglycopeptidase of the oligosaccharides from CAMPATH-1H (a recombinanthumanized murine monoclonal IgG1 antibody which recognizes the CDw52antigen of human lymphocytes) produced in Chinese Hamster Ovary (CHO)cells resulted in a complete reduction in complement mediated lysis(CMCL) (Boyd et al., Mol. Immunol. 32:1311-1318 (1996]), while selectiveremoval of sialic acid residues using neuraminidase resulted in no lossof DMCL. Glycosylation of antibodies has also been reported to affectantibody-dependent cellular cytotoxicity (ADCC). In particular, CHOcells with tetracycline-regulated expression ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GlcNAc, wasreported to have improved ADCC activity (Umana et al., Mature Biotech.17:176-180 [1999]).

Glycosylation variants of antibodies are variants in which theglycosylation pattern of an antibody is altered. By altering is meantdeleting one or more carbohydrate moieties found in the antibody, addingone or more carbohydrate moieties to the antibody, changing thecomposition of glycosylation (glycosylation pattern), the extent ofglycosylation, etc. Glycosylation variants may, for example, be preparedby removing, changing and/or adding one or more glycosylation sites inthe nucleic acid sequence encoding the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

The glycosylation (including glycosylation pattern) of antibodies mayalso be altered without altering the underlying nucleotide sequence.Glycosylation largely depends on the host cell used to express theantibody. Since the cell type used for expression of recombinantglycoproteins, e.g. antibodies, as potential therapeutics is rarely thenative cell, significant variations in the glycosylation pattern of theantibodies can be expected (see, e.g. Hse et al., J. Biol. Chem.272:9062-9070 (1997]). In addition to the choice of host cells, factorswhich affect glycosylation during recombinant production of antibodiesinclude growth mode, media formulation, culture density, oxygenation,pH, purification schemes and the like. Various methods have beenproposed to alter the glycosylation pattern achieved in a particularhost organism including introducing or overexpressing certain enzymesinvolved in oligosaccharide production (U.S. Pat. Nos. 5,047,335;5,510,261 and 5,278,299). Glycosylation, or certain types ofglycosylation, can be enzymatically removed from the glycoprotein, forexample using endoglycosidase H (Endo H). In addition, the recombinanthost cell can be genetically engineered, e.g. make defective inprocessing certain types of polysaccharides. These and similartechniques are well known in the art.

The glycosylation structure of antibodies can be readily analyzed byconventional techniques of carbohydrate analysis, including lectinchromatography, NMR, Mass spectrometry, HPLC, GPC, monosaccharidecompositional analysis, sequential enzymatic digestion, and HPAEC-PAD,which uses high pH anion exchange chromatography to separateoligosaccharides based on charge. Methods for releasing oligosaccharidesfor analytical purposes are also known, and include, without limitation,enzymatic treatment (commonly performed using peptide-N-glycosidaseF/endo-β-galactosidase), elimination using harsh alkaline environment torelease mainly O-linked structures, and chemical methods using anhydroushydrazine to release both N- and O-linked oligosaccharides.

Triabodies are also within the scope of the invention. Such antibodiesare described for instance in Iliades et al., supra and Kortt et al.,supra.

The antibodies of the present invention may be modified by conjugatingthe antibody to a cytotoxic agent (like a toxin molecule) or aprodrug-activating enzyme which converts a prodrug (e.g. a peptidylchemotherapeutic agent, see WO81/01145) to an active anti-cancer drug.See, for example, WO 88/07378 and U.S. Pat. No. 4,975,278. Thistechnology is also referred to as “Antibody Dependent Enzyme MediatedProdrug Therapy” (ADEPT).

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form. Enzymes that are useful in themethod of this invention include, but are not limited to, alkalinephosphatase useful for converting phosphate-containing prodrugs intofree drugs; arylsulfatase useful for converting sulfate-containingprodrugs into free drugs; cytosine deaminase useful for convertingnon-toxic 5-fluorocytosine into the anti-cancer drug, 5-fluorouracil;proteases, such as serratia protease, thermolysin, subtilisin,carboxypeptidases and cathepsins (such as cathepsins B and L), that areuseful for converting peptide-containing prodrugs into free drugs;caspases such as caspase-3; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as beta-galactosidase andneuraminidase useful for converting glycosylated prodrugs into freedrugs; beta-lactamase useful for converting drugs derivatized withbeta-lactams into free drugs; and penicillin amidases, such aspenicillin V amidase or penicillin G amidase, useful for convertingdrugs derivatized at their amine nitrogens with phenoxyacetyl orphenylacetyl groups, respectively, into free drugs. Alternatively,antibodies with enzymatic activity, also known in the art as “abzymes”,can be used to convert the prodrugs of the invention into free activedrugs (see, e.g., Massey, Nature 328: 457-458 (1987)). Antibody-abzymeconjugates can be prepared as described herein for delivery of theabzyme to a tumor cell population.

The enzymes can be covalently bound to the antibodies by techniques wellknown in the art such as the use of heterobifunctional crosslinkingreagents. Alternatively, fusion proteins comprising at least the antigenbinding region of an antibody of the invention linked to at least afunctionally active portion of an enzyme of the invention can beconstructed using recombinant DNA techniques well known in the art (see,e.g., Neuberger et al., Nature, 312: 604-608 (1984).

Further antibody modifications are contemplated. For example, theantibody may be linked to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, orcopolymers of polyethylene glycol and polypropylene glycol, or othermolecules such as polyglutamate. The antibody also may be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Osol, A., Ed.,(1980). To increase the serum half life of the antibody, one mayincorporate a salvage receptor binding epitope into the antibody(especially an antibody fragment) as described in U.S. Pat. No.5,739,277, for example. As used herein, the term “salvage receptorbinding epitope” refers to an epitope of the Fc region of an IgGmolecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that is responsible forincreasing the in vivo serum half-life of the IgG molecule.

Formulations

The WISP-1 antagonists described herein, are optionally employed in acarrier. Suitable carriers and their formulations are described inRemington's Pharmaceutical Sciences, 16th ed., 1980, Mack PublishingCo., edited by Osol et al. Typically, an appropriate amount of apharmaceutically-acceptable salt is used in the carrier to render theformulation isotonic. Examples of the carrier include saline, Ringer'ssolution and dextrose solution. The pH of the carrier is preferably fromabout 5 to about 8, and more preferably from about 7.4 to about 7.8. Itwill be apparent to those persons skilled in the art that certaincarriers may be more preferable depending upon, for instance, the routeof administration and concentration of active agent being administered.The carrier may be in the form of a lyophilized formulation or aqueoussolution.

Acceptable carriers, excipients, or stabilizers are preferably nontoxicto cells and/or recipients at the dosages and concentrations employed,and include buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.

The antagonists described herein may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration should besterile. This is readily accomplished by filtration through sterilefiltration membranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the active agent, which matrices are inthe form of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

Modes of Therapy

The molecules described herein are useful in treating variouspathological conditions, such as cancer. These conditions can be treatedby administration of one or more antagonists described herein.

Diagnosis in mammals of the various pathological conditions describedherein can be made by the skilled practitioner. Diagnostic techniquesare available in the art which allow, e.g., for the diagnosis ordetection of cancer or immune related disease in a mammal. For instance,cancers may be identified through techniques, including but not limitedto, palpation, blood analysis, x-ray, NMR and the like.

The antagonist(s) can be administered in accord with known methods, suchas intravenous administration as a bolus or by continuous infusion overa period of time, by intramuscular, intraperitoneal, intracerebrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Optionally, administration may beperformed through mini-pump infusion using various commerciallyavailable devices. The antagonists may also be employed using genetherapy techniques which have been described in the art.

Effective dosages and schedules for administering antagonists may bedetermined empirically, and making such determinations is within theskill in the art. Single or multiple dosages may be employed. It ispresently believed that an effective dosage or amount of antagonist usedalone may range from about 1 μg/kg to about 100 mg/kg of body weight ormore per day. Interspecies scaling of dosages can be performed in amanner known in the art, e.g., as disclosed in Mordenti et al.,Pharmaceut. Res., 8:1351 (1991).

When in vivo administration of an antagonist thereof is employed, normaldosage amounts may vary from about 10 ng/kg to up to 100 mg/kg of mammalbody weight or more per day, preferably about 1 μg/kg/day to 10mg/kg/day, depending upon the route of administration. Guidance as toparticular dosages and methods of delivery is provided in theliterature; see, for example, U.S. Pat. No. 4,657,760; 5,206,344; or5,225,212. It is anticipated that different formulations will beeffective for different treatment compounds and different disorders,that administration targeting one organ or tissue, for example, maynecessitate delivery in a manner different from that to another organ ortissue. Those skilled in the art will understand that the dosage ofantagonist that must be administered will vary depending on, forexample, the mammal which will receive the agonist or antagonist, theroute of administration, and other drugs or therapies being administeredto the mammal.

Depending on the type of cells and/or severity of the disease, about 1μg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of antagonist antibody is aninitial candidate dosage for administration, whether, for example, byone or more separate administrations, or by continuous infusion. Atypical daily dosage might range from about 1 μg/kg to 100 mg/kg ormore, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful.

A single type of antagonist may be used in the methods of the invention.For example, a WISP-1 immunoadhesin molecule may be administered.Alternatively, the skilled practitioner may opt to employ a combinationof antagonists in the methods, e.g., a combination of a WISP-1immunoadhesin and WISP-1 antibody. It is contemplated that yetadditional therapies may be employed in the methods. The one or moreother therapies may include but are not limited to, administration ofradiation therapy, cytokine(s), growth inhibitory agent(s),chemotherapeutic agent(s), cytotoxic agent(s), tyrosine kinaseinhibitors, ras farnesyl transferase inhibitors, angiogenesisinhibitors, and cyclin-dependent kinase inhibitors which are known inthe art and defined further with particularity in Section I above. Inaddition, therapies based on therapeutic antibodies that target tumorantigens such as Rituxan™ or Herceptin™ as well as anti-angiogenicantibodies such as anti-VEGF.

Preparation and dosing schedules for chemotherapeutic agents may be usedaccording to manufacturers' instructions or as determined empirically bythe skilled practitioner. Preparation and dosing schedules for suchchemotherapy are also described in Chemotherapy Service Ed., M. C.Perry, Williams & Wilkins, Baltimore, Md. (1992). The chemotherapeuticagent may precede, or follow administration of, e.g. an antagonist, ormay be given simultaneously therewith. The antagonist, for instance, mayalso be combined with an anti-oestrogen compound such as tamoxifen or ananti-progesterone such as onapristone (see, EP 616812) in dosages knownfor such molecules.

It may be desirable to also administer antibodies against otherantigens, such as antibodies which bind to CD20, CD11a, CD18, CD40,CD44, ErbB2, EGFR, ErbB3, ErbB4, vascular endothelial factor (VEGF), orother TNFR family members (such as DR4, DR5, OPG, TNFR1, TNFR2).Alternatively, or in addition, two or more antibodies binding the sameor two or more different antigens disclosed herein may beco-administered to the patient. Sometimes, it may be beneficial to alsoadminister one or more cytokines to the patient. In one embodiment, theantagonists herein are co-administered with a growth inhibitory agent.For example, the growth inhibitory agent may be administered first,followed by an antagonist of the present invention.

The antagonist and one or more other therapies may be administeredconcurrently or sequentially. Following administration of antagonist,treated cells in vitro can be analyzed. Where there has been in vivotreatment, a treated mammal can be monitored in various ways well knownto the skilled practitioner.

Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the disorders describedabove is provided. The article of manufacture comprises a container anda label. Suitable containers include, for example, bottles, vials,syringes, and test tubes. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is effective for treating the condition and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). The active agents in the composition may compriseantagonist(s). The label on, or associated with, the container indicatesthat the composition is used for treating the condition of choice. Thearticle of manufacture may further comprise a second containercomprising a pharmaceutically-acceptable buffer, such asphosphate-buffered saline, Ringer's solution and dextrose solution. Itmay further include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va. Unless otherwise noted, thepresent invention uses standard procedures of recombinant DNAtechnology, such as those described hereinabove and in the followingtextbooks: Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press N.Y., 1989; Ausubel et al., Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience,N.Y., 1989; Innis et al., PCR Protocols: A Guide to Methods andApplications, Academic Press, Inc., N.Y., 1990; Harlow et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor, 1988; Gait, M. J., Oligonucleotide Synthesis, IRL Press, Oxford,1984; R. I. Freshney, Animal Cell Culture, 1987; Coligan et al., CurrentProtocols in Immunology, 1991.

A description of the assays and materials referred to in the followingExamples is provided below:

Proteins and Antibodies: Full length human WISP-1-Fc fusion protein wasexpressed and purified as described previously (Desnoyers et al., J.Biol. Chem., 276:47599-47607 (2001)). Purified mouse anti-rat CD44antibody (clone OX49) was from BD Bioscience (Bedford Mass.). The mouseIgG2a isotype control antibody was from Pharmingen. The FITC conjugatedmouse anti-rat CD44 was from Serotec (Raleigh, N.C.) and the FITCconjugated mouse IgG1 isotype control antibody was from DAKO (Denmark).The rat anti-mouse CD44 (clone KM114) was from Pharmingen. The actinantibody (clone C4) was from ICN biomedicals (Aurora, Ohio). Murineanti-human WISP-1 monoclonal antibodies were generated and selectedusing WISP-1-Fc as an immunogen and the protocols and reagents describedin the Examples of U.S. Pat. No. 6,387,657 issued May 14, 2002. Fivesuch monoclonal antibodies have been deposited by Applicants with theATCC, as noted below.

Cells and Tissue Specimens: NRK-49F and SW480 cell lines (ATCC,Manassas, Va.) were maintained in High glucose Dulbecco's ModifiedEagle's Medium (HGDMEM) supplemented with 10% fetal calf serum (FBS).Parental C57MG, C57MG expressing Wnt-1 (C57MG/Wnt-1), NRK-49F expressinghigh levels of hWISP-1 (NRK/WISP-1H), NRK-49F expressing lower levels ofhWISP-1 (NRK/WISP-1L) and NRK-49F containing an empty vector(NRK/control) were obtained from Arnold Levine (Princeton University,Princeton, N.J.) and maintained in media containing 2.5 μg ml⁻¹puromycin (Xu et al., Genes Dev., 14:585-595 (2000)). Wnt-1 transgenicmice obtained from Harold Varmus (National Cancer Institute, NationalInstitutes of Health, Bethesda, Md.) were bred with p53 null miceobtained from Jackson Laboratories (Bar Harbor, Mass.) to generate theWnt-1 trangenic/p53 knock out mice. Specimens of breast tumor andmammary duct epithelium were excised from those mice for analysis.

Particle Exclusion Assay: The erythrocyte sedimentation assay was usedas described previously, with minor modifications. (Knudson et al., J.Cell Sci., 99:227-235 (1991)). NRK/WISP-1 ((normal rat kidneyfibroblasts transfected with WISP-1; see Xu et al., Genes Dev.,14:585-595 (2000)) and NRK/control cells (normal rat kidney fibroblasts;obtained from Arnold Levine, Princeton University, Princeton, N.J.)(1×10⁵ cells/well) were seeded in Falcon 6 well plates (BectonDickinson, Franklin Lanes, N.J.) and maintained in high glucoseDulbecco's modified Eagle's Medium supplemented with 10% fetal bovineserum (FBS) (Life Technologies, Gaithersburg, Md.) overnight at 37° C.,5% CO₂. The next day, the media was removed and 10⁸ sheep RBCs(Inter-Cell Technologies, Hopewell, N.J.) in 750 μl PBS/0.1% BSA(Boehringer Mannheim, Indianapolis, Ind.) were added and allowed tosettle for 15 minutes. The cells were observed using a Nikon Diaphot 300inverted microscope, and digital images were acquired using a SONYDKC-5000 digital photo camera (Japan).

Hyaluronan Staining of Cultured Cells: Hyaluronan

Staining was performed as previously described, with minor modifications(Pienimaki et al., J. Biol. Chem., 276:20428-20435 (2001)). NRK/WISP-1and NRK/control cells (8-10×10³) were plated in 8 well plastic chamberslides and maintained (as described above) overnight at 37° C., 5% CO₂.The next day the cells were washed with phosphate buffered saline (PBS)and fixed with 4% paraformaldehyde in PBS for 20 minutes at roomtemperature. The cells were washed with PBS and the non-specific bindingsites were saturated for 30 minutes with distilled water containing 1%bovine serum albumin (BSA) and 0.1% Triton X-100 (Calbiochem, LaJolla,Calif.). Biotinylated HA binding protein (HA-BP) (Seikagaku America,Falmouth, Mass.) (5 μg/ml in PBS/3% BSA) was added and incubatedovernight at room temperature. The next day the cells were washed withPBS and incubated for 30 minutes with 1:1000 FITC-conjugatedstreptavidin (DAKO, Carpinteria, Calif.) in TBS and 1%/BSA. The cellswere washed in PBS, mounted using Vectashield (Burlingame, Calif.)containing 1 μg/ml Hoechst 33342 (Molecular Probes, Eugene, Oreg.) andvisualized under a Nikon Eclipse 800 fluorescent microscope. Images wereacquired with a Photometrics 300 CCD camera operated at −40° C.interfaced with an Apple G3 computer. The same procedure was also usedto stain frozen sections of mammary tumors from Wnt-1 transgenic mice).

Real time RT-PCR: Relative RNA expression level was determined usingreal-time RT-PCR as previously reported (Winer et al., Anal. Biochem.,270:41-49 (1999)). Total RNA was extracted from cells with Tri-Reagentusing the manufacturer's protocol (Molecular Research Center). Specificprimers and fluorogenic probes were designed and used to amplify andquantitate gene expression level. The genes' specific signals werenormalized to that of the glyceraldehyde-3-phosphate dehydrogenasehousekeeping gene. Triplicate sets of data were averaged for eachcondition. All TaqMan RT-PCR reagents were purchased from AppliedBiosystems (Foster City, Calif.). To test the effect of recombinantWISP-1 on gene expression, petri dishes (35 mm) were coated overnight at4° C. with 75 μg ml⁻¹ hWISP-1-Fc in PBS. The next day, cells (3×10⁵cells/dish) were plated on the coated surface and the expressionanalysis was performed after 18 hours. In certain cases the coatedsurface was washed and treated for an additional 2 hours at roomtemperature before cell were seeded.

FACS Analysis: Cells were harvested by trypsinization, washed with PBSand incubated in PBS 1% BSA/20% glucose containing 1 μg ml⁻¹ FITCconjugated anti-CD44 (Serotec, Raleigh, N.C.) or an isotype control Ab(DAKO, Denmark) for 30 minutes at room temperature. Cells were washedwith the same buffer and analyzed on an EPICS XL-MCL (Coulter, Miami,Fla.).

Western Blot: NRK/C and NRK/W1H were cultured to confluency in 10 cm²plates. The cells were washed with PBS and extracted with 300 μl of 1%Triton-X100 in TBS for 5 minutes. Proteins were precipitated by adding10 volumes of acetone to the extract. Samples were reduced, denaturedand loaded on a 4-20% gradient SDS polyacrylamide gel. The gel wastransferred to PVDF membranes and probed with a anti-rat CD44 antibody(OX49) or an anti-mouse CD44 (KM114) at a 1 μg/ml concentration anddetected using a HRP-conjugated anti-mouse IgG secondary antibody andthe Amersham ECL detection reagents. Actin detection in the samples wasused as loading control.

Scatter Assay: The cells were plated in 24 well plates at lowconcentration (˜1000 cells/well) and allowed to form colonies over 3days. The cells were stained using Diff-Quik (Dade Behring, Newark,Del.) and colonies were observed under the microscope.

Cell Wound Healing Assay: Cell migration was evaluated using a cellwound healing assay previously described with minor modifications(Pienimaki et al., J. Biol. Chem., 276:20428-20435 (2001)). Briefly,cells were cultured until they reached confluency. One line (˜1 mm) wasdrawn in the cell layer using a micropipette equipped with a disposableyellow tip. Cell migration over the cleared area was monitored by timelapse microscopy over 15 hours. The area newly occupied by the cellsafter this period was measured using NIH image software calibrated witha stage micrometer.

Time Lapse Microscopy: Cells were plated in HGDMEM/10% FBS and theirrandom migration was observed for 15 hours using time lapse microscopy.The distance covered by the cells was measured using NIH image softwarecalibrated with a stage micrometer.

Migration Assay: Haptotaxis was measured using a modified Boyden chambersystem as described previously (Bourguignon et al., J. Biol. Chem.,275:1829-1838 (2000)). The underside of 8 μm porosity 24-well format PETmembrane filters (Falcon) were coated overnight at 4° C. with 50 μl ofprotein (50 μg ml⁻¹) in PBS. The coated inserts were rinsed inserum-free media and 0.5×10⁵ NRK cells in 0.5 ml HGDMEM/10% FBS wereadded to the upper chamber. The lower chamber was filled with 0.75 ml ofthe same media and the plates were incubated overnight at 37° C. Thenext day the upper chamber was wiped with a cotton swab and the cellsthat migrated to the lower side of the insert were stained with theDiff-Quik Stain Kit (Dade Behring Inc.) and counted under themicroscope. Triplicate sets of data were averaged for each condition.

In certain cases, the coated inserts were washed and treated for anadditional 2 hours at room temperature.

In situ Hybridization: PCR primers were designed to amplify either a 740bp fragment of murine WISP-1 spanning from nt 440-1180 of NM_(—)018865(upper-5′ GGCTGCCATCTGTGACCCA (SEQ ID NO:12) and lower-5′CATAGGACCTGCCGGGAGAA A (SEQ ID NO:13)) or a 706 bp fragment of murine WISP-1spanning from nt 204-910 of NM_(—)018865 (upper-5′ GCCGTGGCAGTCCTGAGGG(SEQ ID NO:14) and lower-5′CAGCACCGGG CATTGACGTT A (SEQ ID NO:15)) or a464 bp fragment of murine CD44 spanning from nt 144-608 of M27129(upper-5′ TGGAGAAAAATGGCCGCTAC A (SEQ ID NO:16)) and lower-5′ TGGGGTGCTCTTCTCGATGG (SEQ ID NO:17)) or a 630 bp fragment of murine HAS2 spanningfrom nt 927-1557 of NM_(—)008216 (upper-5′ GGACAAATCGGCCACGTACA T (SEQID NO:18) and lower-5′CTTGCTCCAT CGGGTCTGC (SEQ ID NO:19)). Primersincluded extensions encoding 27-nucleotide T7 or T3 RNA polymeraseinitiation sites to allow in vitro transcription of sense or antisenseprobes, respectively, from the amplified products. (Lu et al., CellVision, 1:169-176 (1994)). All tissues were fixed in 4% formalin andparaffin-embedded. Sections 3-5 microns thick were deparaffinized,deproteinated in 4 mg/ml of proteinase K for 30 minutes at 37° C., andfurther processed for in situ hybridization as previously described.(Holcomb et al., EMBO J., August 2000 1;19(15):4046-55). ³³P-UTP labeledsense and antisense probes were hybridized to the sections at 55° C.overnight. Unhybridized probe was removed by incubation in 20 mg/mlRNase A for 30 minutes at 37° C., followed by a high stringency wash at55° C. in 0.1×SSC for 2 hours and dehydration through graded ethanols.The slides were dipped in NBT2 nuclear track emulsion, exposed in sealedplastic slide boxes containing dessicant for 4 weeks at 4° C., developedand counterstained with hematoxylin and eosin.

Example 1

Stable fibroblast cell lines expressing high (NRK/WISP-1H) or low(NRK/WISP-1L) levels of WISP-1 and a control cell line containing anempty vector (NRK/control) were used to evaluate the effect of WISP-1 onHA production. As demonstrated by the particle exclusion assay,NRK/WISP-1H cells accumulated a large hyaluronan pericellular coatcapable of excluding sedimenting erythrocytes (FIG. 1 a). NRK/WISP-1Laccumulated a smaller pericellular matrix (FIG. 1 b) whereas no matrixsurrounded NRK/control (FIG. 1 c) or NRK/WISP-1H cells treated withhyaluronidase (data not shown). Accumulation of cell surface associatedHA was also evaluated by fluorescent staining using the biotinylated HAbinding protein (bHABP). The staining revealed HA accumulation at theNRK/WISP-1H cell surface (FIG. 1 d) whereas no staining was detected onNRK control cells (FIG. 1 e).

The effect of WISP-1 on HA secretion was evaluated by comparing theaccumulation of HA in the culture media of NRK/WISP-1H to NRK/controlcells over time. After 24 hours, HA concentration in the NRK/WISP-1Hmedia was 3.5 fold greater in NRK/control cell media and graduallyincreased to 8 fold after 144 h (FIG. 1 f). Together these results showthat WISP-1 expression in NRK cells promotes HA accumulation at the cellsurface and in the culture media.

Example 2

To identify the enzyme responsible for the WISP-1 triggered HAproduction, the expression of the known HA synthases (HAS1, HAS2 andHAS3) in NRK/WISP-1H, NRK/WISP-1L and NRK/control cells was analyzed.HAS2 expression was increased up to 10 fold in WISP-1 producing cellswhereas HAS1 and HAS3 mRNA levels were identical to the control (FIG. 2a). CD44 and RHAMM mRNA levels were also increased in NRK/WISP-1 celllines whereas hyaluronidase expression remained unchanged (FIG. 2 a).Moreover, HAS2, CD44 and RHAMM mRNA levels were proportional to WISP-1expression. As demonstrated by FACS analysis (FIG. 2 b) and Western blot(FIG. 2 c), the increase in CD44 mRNA expression in the NRK/WISP-1Hcells resulted in a 4-fold increase in CD44 protein level.

Example 3

HA was shown to stimulate cell migration by interacting with two cellsurface receptors, CD44 and RHAMM (Hall et al., supra; Bourguignon etal., supra). Because WISP-1 increased both HA production and CD44 andRHAMM expression, the motility of WISP-1 expressing cells was evaluated.When plated at a low density, NRK/control cells proliferated and formedwell defined colonies (FIG. 3 a). Proliferating NRK/WISP-1L cells formedless defined groups as some cells departed from the growing colonies(FIG. 3 b). No NRK/WISP-1H colonies were seen, cells scattered in arandom pattern (FIG. 3 c). Contrary to the control (FIG. 3 d), WISP-1expressing cells also revealed a hyper elongated morphology withextended lamellipodia characteristic of high motility (FIG. 3 e). Usingtime lapse microscopy, a 4 fold increase in the migration distance ofNRK/WISP-1H was observed compared to control cells (FIG. 3 f).NRK/WISP-1H also showed a 2.5 fold increase in migration area in a cellwound healing assay (FIG. 3 g). Together these results demonstrate thatWISP-1 expression may promote cell migration.

Example 4

To determine whether the ectopic addition of WISP-1 could promote HAS2expression and cell migration, an assay was conducted using purifiedrecombinant WISP-1-IgG (Desnoyers et al., supra). WISP-1 addition to theculture media did not promote HAS2 expression (data not shown). When NRKcells were plated onto a WISP-1-IgG coated surface, HAS2 expressionincreased compared to cells plated onto uncoated plastic or onto anirrelevant IgG chimeric protein (TNFR-IgG) coated surface (FIG. 4 a).Although decorin alone promoted HAS2 expression, this induction wasfurther increased three fold after the coated surface was incubated withWISP-1. The effects of ectopic WISP-1 addition on NRK cell migrationwere also examined. In a transwell assay, WISP-1-IgG induced thehaptotactic migration of NRK cells when coated on the filter's lowersurface (FIG. 4 b). Coating of an irrelevant IgG chimeric protein(TNFR-IgG) or addition of WISP-1-IgG to the lower chamber did notpromote migration. A CD44 or a WISP-1 antibody (FIG. 4 b) inhibited thehaptotactic migration induced by WISP-1. Moreover, WISP-1 haptotacticactivity was not limited to fibroblasts as it also promoted themigration of SW480 cells, a colon adenocarcinoma cell line (FIG. 4 c).Together these results demonstrate that ectopic WISP-1 additionincreases HAS2 expression and promotes cell migration through a CD44mediated mechanism. It is believed, though not fully understood, thatWISP-1 presentation may be important for migration as it may need to betethered to a substrate to elicit this activity.

Example 5

Because WISP-1 is believed to be a downstream effector of Wnt-1, WISP-1,HAS2 and CD44 expression in a mammary epithelial cell line stablytransfected with Wnt-1 (C57MG/Wnt-1) were analyzed. When compared to thecontrol cell line (C57MG), C57MG/Wnt-1 cells showed a 2.7, 5.8 and 3fold increase in WISP-1, HAS2 and CD44 mRNA expression respectively(FIG. 5 a). As demonstrated by Western blot, C57/Wnt-1 cells also showeda 6-fold increase in CD44 protein content (FIG. 5 b). Moreover, unlikethe control cell line, Wnt-1 expressing cells failed to form distinctcolonies and scattered when put in culture at low density (FIG. 5 c).These results show that HAS2 and CD44 expression and cell motility areelevated in cell lines where WISP-1 expression is triggered by Wnt-1.

Example 6

Wnt-1 expression in mammary epithelium promotes tumor development intransgenic mice (Li et al., Oncogene, 19:1002-1009 (2000); Tsukamoto etal., Cell, 55:619-625 (1988)). Because WISP-1 is a putative downstreameffector of Wnt-1, the mRNA expression of HAS2 and CD44 in spontaneousmammary tumors from MMTV-Wnt-1 trangenic mice was measured. Whencompared to mammary duct epithelium, HAS2 mRNA expression was increasedbetween 2.5 and 5.5 fold in all mammary tumors analyzed (n=5; FIG. 5 d).Similarly, CD44 mRNA expression was induced between 2.2 and 4.2 fold inall tumors (n=5; FIG. 5 e). These results show that HAS2 and CD44 areoverexpressed in MMTV-Wnt-1 trangenic mice mammary tumors expressingWISP-1.

Example 7

In situ hybridization demonstrated elevated WISP-1 expression in theperitumoral stroma of MMTV-Wnt-1 transgenic mouse mammary tumors (FIG. 6a, b). In contrast, HAS2 (FIG. 6 c-d) and CD44 (FIG. 6 e-f) expressionwas found only in tumoral epithelial cells. The localization of CD44 inthe tumor parenchyma was confirmed by immunohistochemistry (FIG. 6 g).Link protein staining revealed an accumulation of hyaluronan associatedwith the tumor stroma (FIG. 6 h). The highest staining intensity wasfound in the immediate vicinity of the tumor lobule whereas weakerstaining was localized to the normal mammary duct epithelium. Thesefindings are consistent with the suggestion that WISP-1 may regulateinteractions between tumor and stromal cells that involve hyaluronicacid and CD44.

Example 8

The metastasis and growth potential of transformed lines was evaluatedas described previously (Welch et al., Cancer Res., 60:1552-1556(2000)). Nine-week-old Swiss nu/nu female mice were used. Briefly, cellswere harvested by trypsinization and washed twice with PBS. Each mousewas injected into the lateral tail vein with 100 μl of a suspensioncontaining 5×10⁴ or 2.5×10⁵ cells. At 2, 3 and 4 week postinjection,mice were examined using cine-magnetic resonance imaging (MRI) forapparition of pulmonary lesions and necropsies were performed. The lungswere perfused in Bouin's fixative, excised and H&E stained sections weregenerated for evaluation of pulmonary tumor colonization.

Longitudinal sections of the left lung, and a single transverse sectionof the cranial, medial, caudal, and accessory lobes of the right lungwere evaluated. Histologically, affected lungs had spindloid neoplasticcells that infiltrated the pulmonary interstitium. However, because theseverity of the changes was variable the following grading system wasestablished.

I=Minimal involvement of the pulmonary interstitium—10-20% of the lungis affected. Nests and clusters of spindloid neoplastic cellsmultifocally disrupt the pulmonary interstitium. Affected foci arecommonly along the pleural surface, extending into the interstitium. Novessels or large airways are affected.

II=Moderate involvement of the pulmonary interstitium—20-50% of the lungis affected. Nests and clusters of spindloid neoplastic cellsmultifocally disrupt the pulmonary interstitium. Some vessels or largeairways are filled with spindloid cells. Spindloid cells frequently forma broad band or large mass subjacent to the pleura and extending intothe interstitium. Overlying mesothelium is plump (reactive).

III=Severe involvement of the pulmonary interstitium—50-100% of the lungis affected. Nests and clusters of spindloid neoplastic cellsmultifocally disrupt the pulmonary interstitium. Blood vessels and largeairways are filled with spindloid cells. Spindloid cells form a broadband or large mass subjacent to the pleura and extending into theinterstitium. Spindloid cells are often embedded in a pale basophilic toamphophilic acellular material. Overlying mesothelium is plump(reactive). Minimal unaffected area for gas exchange remains.

The results of the lung colonization of nude mice inoculated withNRK/WISP-1H, NRK/WISP-1L and NRK/control cells are summarized in FIGS. 7and 10. In mice inoculated with NRK/WISP-1H or NRK/WISP-1L, cells formedlung masses in a time- and dose-dependent manner. The most severelesions were seen in mice injected with 2.5×10⁵ NRK/WISP-1H andnecropsied at 4 weeks post-injection. At 2 and 3 weeks post-injection,the mice had less severe but progressive tumor formation (grade I-II;see grading system above). Mice injected with 2.5×10⁵ NRK/WISP-1L hadonly minimal lung tumor formation by 4 weeks post-injection. After 4weeks post-injection, mice inoculated with 0.5×10⁵ NRK/WISP-1L cellswere either normal or had minimal neoplastic infiltration (grade I),whereas mice injected with 0.5×10⁵ NRK/WISP-1H cells showed increasedneoplastic infiltration (grade I-II). Histological observation revealedthat the lungs of animals inoculated with NRK/control cells were normal(FIG. 7 a-c). In NRK/WISP-1 injected animals, neoplastic spindloid cellsinitially formed small clusters within, protruding from, or subjacent tothe pleural surface of the lungs (grade I; FIG. 7 d-f) often embedded ina pale basophilic to amphophilic acellular material. In more severelyaffected animals, the neoplastic cells formed a broad confluent bandsubjacent to the pleura (grade II; FIG. 7 g-i). In the most severelyaffected animals, blood vessels and large airways were filled withspindloid cells. (grade III; FIG. 7 j-l). MRI analysis showed increaseddensity (hypersignal) outlining the right and left lungs along thepleura in animals with histologic grade II and grade III invasion.Severity of the lesion on MRI was consistent with the histologic score(FIG. 7 a, d, g, j). These results indicate that WISP-1 expressionpromotes cellular metastatic growth potential.

Example 9

NRK/WISP-1H cells (2.5×10⁵ cells) were inoculated in the tail vein ofnude mice. Starting on the day of the cell inoculation, the mice wereinjected intraperitoneally twice a week with 10 mg/kg of a CD44antibody, an isotype control antibody or with buffer only (PBS). Thelungs were fixed and excised after four weeks for gross anatomicalanalysis.

After 4 weeks, the severity of the lesions found in CD44 antibodytreated animals (n=5) varied from normal to grade I whereas all (n=10)animals treated with a control antibody or saline had grade III lesions(FIG. 8 a). The average area of metastatic foci in the lungs of animalstreated with CD44 antibody was reduced by 99% (P<0.00003) compared toanimals treated with a control antibody (FIG. 8 b).

Example 10

An assay was conducted to examine binding specificity of certain WISP-1antibodies. Full length mouse WISP-1 (GenBank accession numberNM_(—)018865) and full length human WISP-1 (GenBank accession numberAF100779; FIG. 9) were cloned into an expression vector encoding thehuman IgG₁ Fc region downstream of the WISP-1 sequence. The resultingrecombinant fusion protein (WISP-1-Fc) was synthesized in a baculovirusexpression system using Sf9 insect cells and purified to homogeneityfrom serum-free conditioned medium by affinity chromatography on aProtein A-Sepharose 4 Fast Flow (Amersham Pharmacia Biotech). Fulllength human WISP-1 was also expressed with an amino terminalhexa-histidine tag (WISP-1-His) in an E. coli strain. The cell lysatewas subjected to chromatography on a Ni²⁺-NTA agarose column (Qiagen).WISP-1-His was eluted with a 0 to 500 mM imidazole gradient. Fractionscontaining the eluted WISP-1-His were then pooled and dialyzed. HumanWISP-1 from a mammalian expression system was obtained by lysing NRKcells stably transfected with human WISP-1 (Arnold Levine; PrincetonUniversity, Princeton, N.J.) with SDS-PAGE sample buffer. A control celllysate was generated with NRK cells stably transfected with an emptyvector.

WISP-1 (50 ng) from various expression systems was electrophoresed on aSDS polyacrylamide gel and electro-transferred onto polyvinyldifluoride(PVDF) membranes and probed with different WISP-1 monoclonal antibodies.

WISP-1 antibodies 3D11.D7 (also referred to herein as “3D11”), 11C2.C10(also referred to herein as “11C2”), 9C11.C7 (also referred to herein as“9C11”) and 5D4.F6 (also referred to herein as “5D4”) bound specificallyto WISP-1 generated from baculovirus, bacterial and mammalian expressionsystems (FIG. 11 a). These antibodies did not bind to the murine WISP-1from baculovirus and did not recognize any protein from the controllysate. The WISP-1 antibodies 6F8, 3A7, 10H12, 3A11, 6E3, 3H10, 5G1, and10B1 recognized both human and murine WISP-1 only when generated withthe baculovirus expression system (FIG. 11 b). These antibodies did notrecognize human WISP-1 when produced in a bacterial or mammalianexpression system. The antibody from clone 9C10 did not bind to anyprotein after Western blot.

These results suggest that WISP-1 antibodies 3D11, 11C2, 9C11 and 5D4specifically recognize human WISP-1 and can be used for WISP-1 detectionby Western blot.

Example 11

An assay was conducted to identify the epitopes recognized by the WISP-1antibodies 11C2, 9C11, 5D4 and 3D11.

Full length human WISP-1 (GenBank accession number AF100779) was clonedinto a pIRESpuro2 expression vector (Clontech Laboratories, Palo Alto,Calif.) encoding 6 histidines downstream of the WISP-1 sequence.Deletion mutants were also generated by removing one, two or threedomains of human WISP-1. The resulting contructs were also cloned intothe pIRESpuro2 expression vector. The nomenclature used to identify thedifferent WISP-1 contructs refer to the domains they contain. (see FIG.12B) Domain 1 is the insulin-like growth factor binding protein domain(IFGBP), domain 2 is the von Willebrand factor C (VWFc) domain, domain 3is the thrombospondin (TSP) domain, and the domain 4 is the C-terminal(CT) domain. The variable region resides between domain 2 and 3. Theseregions and domains of WISP-1 are illustrated in FIG. 12A.

The sequences encoding these domains of WISP-1 are as follows:

Sequences of WISP-1 Constructs Domain 1: (SEQ ID NO:3)GAATTCACCATGAGGTGGTTCCTGCCCTGGACGCTGGCAGCAGTGACAGCAGCAGCCGCCAGCACCGTCCTGGCCACGGCCCTCTCTCCAGCCCCTACGACCATGGACTTTACTCCAGCTCCACTGGAGGACACCTCCTCACGCCCCCAATTCTGCAAGTGGCCATGTGAGTGCCCGCCATCCCCACCCCGCTGCCCGCTGGGGGTCAGCCTCATCACAGATGGCTGTGAGTGCTGTAAGATGTGCGCTCAGCAGCTTGGGGACAACTGCACGGAGGCTGCCATCTGTGACCCCCACCGGGGCCTCTACTGTGACTACAGCGGGGACCGCCCGAGGTACGCAATAGGAGTGTGTGCACAGGCGGCCGCACACCACCATCACCATCACCATCACTAAGTGAGGCCGCATAGATAACTGATCCAGTGTGCTGGAATTAATTC Domain 2:(SEQ ID NO:4)GAATTCACCATGAGGTGGTTCCTGCCCTGGACGCTGGCAGCAGTGACAGCAGCAGCCGCCAGCACCGTCCTGGCCACTGCAGTGGTCGGTGTGGGCTGCGTCCTCGATGGGGTGCGCTACAACAACGGCCAGTCCTTCCAGCCTAACTGCAAGTACAACTGCACGTGCATCGACGGCGCGGTGGGCTGCACACCACTGTGCCTCCGAGTGCGCCCCCCGCGTCTCTGGTGCCCCCACCCGCGGCGCGTGAGCATACCTGGCCACTGCTGTGAGCAGTGGGTATGTGCGGCCGCACACCACCATCACCATCACCATCACTAAGTGAGGCCGCATAGATAAC Domain 3: (SEQ ID NO:5)GAATTCACCATGAGGTGGTTCCTGCCCTGGACGCTGGCAGCAGTGACAGCAGCAGCCGCCAGCACCGTCCTGGCCACTGCAGCATGGCACAGGAACTGCATAGCCTACACAAGCCCCTGGAGCCCTTGCTCCACCAGCTGCGGCCTGGGGGTCTCCACTCGGATCTCCAATGTTAACGCCCAGTGCTGGCCTGAGCAAGAGAGCCGCCTCTGCAACTTGCGGCCATGCGATGTGGACATCCATACACTCATTAAGGCGGCCGCACACCACCATCACCATCACCATCACTAAGTGAGGCCGCATAGATAACTGATCCAGTGT Domain 4: (SEQ ID NO:6)GAATTCACCATGAGGTGGTTCCTGCCCTGGACGCTGGCAGCAGTGACAGCAGCAGCCGCCAGCACCGTCCTGGCCACTGCAGGGAAGAAGTGTCTGGCTGTGTACCAGCCAGAGGCATCCATGAACTTCACACTTGCGGGCTGCATCAGCACACGCTCCTATCAACCCAAGTACTGTGGAGTTTGCATGGACAATAGGTGCTGCATCCCCTACAAGTCTAAGACTATCGACGTGTCCTTCCAGTGTCCTGATGGGCTTGGCTTCTCCCGCCAGGTCCTATGGATTAATGCCTGCTTCTGTAACCTGAGCTGTAGGAATCCCAATGACATCTTTGCTGACTTGGAATCCTACCCTGACTTCTCAGAAATTGCCAACGCGGCCGCACACCACCATCACCATCACCATCACTAAGTGAGGCCGCATAGATAACTGATCCAGTGTG Domain1,2: (SEQ ID NO:7)GAATTCACCATGAGGTGGTTCCTGCCCTGGACGCTGGCAGCAGTGACAGCAGCAGCCGCCAGCACCGTCCTGGCCACGGCCCTCTCTCCAGCCCCTACGACCATGGACTTTACTCCAGCTCCACTGGAGGACACCTCCTCACGCCCCCAATTCTGCAAGTGGCCATGTGAGTGCCCGCCATCCCCACCCCGCTGCCCGCTGGGGGTCAGCCTCATCACAGATGGCTGTGAGTGCTGTAAGATGTGCGCTCAGCAGCTTGGGGACAACTGCACGGAGGCTGCCATCTGTGACCCCCACCGGGGCCTCTACTGTGACTACAGCGGGGACCGCCCGAGGTACGCAATAGGAGTGTGTGCACAGGTGGTCGGTGTGGGCTGCGTCCTGGATGGGGTGCGCTACAACAACGGCCAGTCCTTCCAGCCTAACTGCAAGTACAACTGCACGTGCATCGACGGCGCGGTGGGCTGCACACCACTGTGCCTCCGAGTGCGCCCCCCGCGTCTCTGGTGCCCCCACCCGCGGCGCGTGAGCATACCTGGCCACTGCTGTGAGCAGTGGGTATGTGCGGCCGCACACCACCATCACCATCACCATCACTAAGTGAGGCCGCATAGATAAC Domain 1,2,3: (SEQ ID NO:8)GAATTCACCATGAGGTGGTTCCTGCCCTGGACGCTGGCAGCAGTGACAGCAGCAGCCGCCAGCACCGTCCTGGCCACGGCCCTCTCTCCAGCCCCTACGACCATGGACTTTACTCCAGCTCCACTGGAGGACACCTCCTCACGCCCCCAATTCTGCAAGTGGCCATGTGAGTGCCCGCCATCCCCACCCCGCTGCCCGCTGGGGGTCAGCCTCATCACAGATGGCTGTGAGTGCTGTAAGATGTGCGCTCAGCAGCTTGGGGACAACTGCACGGAGGCTGCCATCTGTGACCCCCACCGGGGCCTCTACTGTGACTACAGCGGGGACCGCCCGAGGTACGCAATAGGAGTGTGTCCACAGGTGGTCGGTGTGGGCTGCGTCCTGGATGGGGTGCGCTACAACAACGGCCAGTCCTTCCAGCCTAACTGCAAGTACAACTGCACCTGCATCGACGGCGCGGTGGGCTGCACACCACTGTGCCTCCGAGTGCGCCCCCCGCGTCTCTGGTGCCCCCACCCGCGGCGCGTGAGCATACCTGGCCACTGCTGTGAGCAGTGGGTATGTGAGGACGACGCCAAGAGGCCACGCAAGACCGCACCCCGTGACACAGGAGCCTTCGATGCTGTGGGTGAGGTGGAGGCATGGCACAGGAACTGCATAGCCTACACAAGCCCCTGGAGCCCTTGCTCCACCAGCTGCGGCCTGGGGGTCTCCACTCGGATCTCCAATGTTAACGCCCAGTGCTGGCCTGAGCAAGAGAGCCGCCTCTGCAACTTGCGGCCATGCGATGTGGACATCCATACACTCATTAAGGCgGCCGCACACCACCATCACCATCACCATCACTAAGTGAGGCCGCATAGATAACTGATCCAGTGTGCTGGA Domain 1,2,4: (SEQ IDNO:9)GAATTCACCATGAGGTGGTTCCTGCCCTGGACGCTGGCAGCAGTGACAGCAGCAGCCGCCAGCACCGTCCTGGCCACGGCCCTCTCTCCAGCCCCTACGACCATGGACTTTACTCCAGCTCCACTGGAGGACACCTCCTCACGCCCCCAATTCTGCAAGTGGCCATGTGAGTGCCCGCCATCCCCACCCCGCTGCCCGCTGGGGGTCAGCCTCATCACAGATGGCTGTGAGTGCTGTAAGATGTGCGCTCAGCAGCTTGGGGACAACTGCACGGAGGCTGCCATCTGTGACCCCCACCGGGGCCTCTACTGTGACTACAGCGGGGACCGCCCGAGGTACGCAATAGGAGTGTGTGCACAGGTGGTCGGTGTGGGCTGCGTCCTGGATGGGGTGCGCTACAACAACGGCCAGTCCTTCCAGCCTAACTGCAAGTACAACTGCACGTGCATCGACGGCGCGGTGGGCTGCACACCACTGTGCCTCCGAGTGCGCCCCCCGCGTCTCTGGTGCCCCCACCCGCGGCGCGTGAGCATACCTGGCCACTGCTGTGAGCAGTGGGTATGTCTGCAGGCAGGGAAGAAGTGTCTGGCTGTGTACCAGCCAGAGGCATCCATGAACTTCACACTTGCGGGCTGCATCAGCACACGCTCCTATCAACCCAAGTACTGTGGAGTTTGCATGGACAATAGGTGCTGCATCCCCTACAAGTCTAAGACTATCGACGTGTCCTTCCAGTGTCCTGATGGGCTTGGCTTCTCCCGCCAGGTCCTATGGATTAATGCCTGCTTCTGTAACCTGAGCTGTAGGAATCCCAATGACATCTTTGCTGACTTGGAATCCTACCCTGACTTCTCAGAAATTGCCAACGCGGCCGCACACCACCATCACCATCACCATCACTAAGTGAGGCCGCATAGATAACTGATCCAGTGTGCTGGAATTAATTCGCTGTCTGCGAGGGCCAGCTGTTGGGGTGAGTACTCCCTCTCAAAAGCGGGCATGACTTCTGCGCTA Domain 1,3,4: (SEQ ID NO:10)GAATTCACCATGAGGTGGTTCCTGCCCTGGACGCTGGCAGCAGTGACAGCAGCAGCCGCCAGCACCGTCCTGGCCACCGCCCTCTCTCCAGCCCCTACGACCATGGACTTTACTCCAGCTCCACTGGAGGACACCTCCTCACGCCCCCAATTCTGCAAGTGGCCATGTGAGTGCCCGCCATCCCCACCCCGCTGCCCGCTGGGGGTCAGCCTCATCACAGATGGCTGTGAGTGCTGTAAGATGTGCGCTCAGCAGCTTGGGGACAACTGCACGGAGGCTGCCATCTGTGACCCCCACCGGGGCCTCTACTGTGACTACAGCGGGGACCGCCCGAGGTACGCAATAGGAGTGTGTGCGCATGCTGTGGGTGAGGTGGAGGCATGGCACAGGAACTGCATAGCCTACACAAGCCCCTGGAGCCCTTGCTCCACCAGCTGCGGCCTGGGGGTCTCCACTCGGATCTCCAATGTTAACGCCCAGTGCTGGCCTGAGCAAGAGAGCCGCCTCTGCAACTTGCGGCCATGCGATGTGGACATCCATACACTCATTAAGGCAGGGAAGAAGTGTCTGGCTGTGTACCAGCCAGAGGCATCCATGAACTTCACACTTGCGGGCTGCATCAGCACACGCTCCTATCAACCCAAGTACTGTGGAGTTTGCATGGACAATAGGTGCTGCATCCCCTACAAGTCTAAGACTATCGACGTGTCCTTCCAGTGTCCTGATGGGCTTGGCTTCTCCCGCCAGGTCCTATGGATTAATGCCTGCTTCTGTAACCTGAGCTGTAGGAATCCCAATGACATCTTTGCTGACTTGGAATCCTACCCTGACTTCTCAGAAATTGCCAACGCGGCCGCACACCACCATCACCATCACCATCACTAAGTGAGGCCGCATAGATAACDomain 2,3,4: (SEQ ID NO:11)      GAATTCACCATGAGGTGGTTCCTGCCCTGGACGCTGGCAGCAGTGACAGCAGCAGCCGCCAGCACCGTCCTGGCCACTGCAGTGGTCGGTGTGGGCTGCGTCCTGGATGGGGTGCGCTACAACAACGGCCAGTCCTTCCAGCCTAACTGCAAGTACAACTGCACGTGCATCGACGGCGCGGTGGGCTGCACACCACTGTGCCTCCGAGTGCGCCCCCCGCGTCTCTGGTGCCCCCACCCGCGGCGCGTGAGCATACCTGGCCACTGCTGTGAGCAGTGGGTATGTGAGGACGACGCCAAGAGGCCACGCAAGACCGCACCCCGTGACACAGGAGCCTTCGATGCTGTGGGTGAGGTGGAGGCATGGCACAGGAACTGCATAGCCTACACAAGCCCCTGGAGCCCTTGCTCCACCAGCTGCGGCCTGGGGGTCTCCACTCGGATCTCCAATGTTAACGCCCAGTGCTGGCCTGAGCAAGAGAGCCGCCTCTGCAACTTGCGGCCATGCGATGTGGACATCCATACACTCATTAAGGCAGGGAAGAAGTGTCTGGCTGTGTACCAGCCAGAGGCATCCATGAACTTCACACTTGCGGGCTGCATCAGCACACGCTCCTATCAACCCAAGTACTGTGGAGTTTGCATGGACAATAGGTGCTGCATCCCCTACAAGTCTAAGACTATCGACGTGTCCTTCCAGTGTCCTGATGGGCTTGGCTTCTCCCGCCAGGTCCTATGGATTAATGCCTGCTTCTGTAACCTGAGCTGTAGGAATCCCAATGACATCTTTGCTGACTTGGAATCCTACCCTGACTTCTCAGAAATTGCCAACGCGGCCGCACACCACCATCACCATCACCATCACTAAGTGAGGCCGCATAGATAACTGATCCAGTGTGCTGGAATTAATTCGCTGTCTGCGA

Cells (HEK 293T) were transfected with the different constructs, and theculture media was collected after 48 hours. One milliliter of culturemedia was incubated with 20 μl of cobalt-agarose for 1 hour, centrifugedand washed. The adsorbed proteins were eluted by heating the pellet at100° C. for 5 minutes in 20 μl of SDS-PAGE sample buffer. The sampleswere electrophoresed, electro-transferred onto PVDF and probed with thedifferent WISP-1 antibodies.

Antibodies 11C2, 9C11 and 5D4 recognized only WISP-1 contructscontaining the 19 first amino acids of the variable region locatedbetween domain 2 and 3 (FIGS. 12C; 12E; 12G). The WISP-1 antibody 3D11recognized only WISP-1 contructs containing the domain 1 (amino acids 24to 117; FIGS. 12D; 12F).

These results indicate that the antibodies 11C2, 9C11 and 5D4 recognizespecifically the variable region of WISP-1 whereas the antibody 3D11recognizes specifically the domain 1 of WISP-1.

Example 12

An assay was conducted to identify the epitope recognized by the WISP-1antibody 9C10.F5 (also referred to herein as “9C10”).

Culture media from HEK 293T cells transfected with the various WISP-1deletion contructs (as described above in Example 11) was incubated with1 μg of WISP-1 antibody 9C10 and 20 μl of protein A-agarose for 1 hourat room temperature. The immunocomplex was precipitated bycentrifugation and eluted by heating the pellet at 100° C. for 5 minutesin 20 μl of SDS-PAGE sample buffer. The samples were electrophoresed,electro-transferred onto PVDF and probed with WISP-1 antibody 11C2.

The antibody 9C10 immunoprecipitated only constructs containing thedomain 1 of WISP-1 (FIG. 13). These results demonstrate that theantibody 9C10 specifically recognizes the domain 1 of WISP-1 and can beused for immunoprecipitation.

Example 13

WISP-1 antibody 9C10 (100 μl of 2 μg/ml in carbonate buffer, pH 9.6) wascoated to Maxisorb plates overnight at 4° C. The plates were blockedwith 200 μl of PBS/3% BSA for 1 hour. A standard curve was made ofserial dilutions of WISP-1-Fc (100 μl in PBS/3% BSA) and incubated for 1hour. After the incubation, the plates were washed with 100 μl PBS/0.05%Tween and WISP-1 antibodies (100 μl of 2 μg/ml) in PBS/3% BSA(biotinylated 11C2 or 55B) were incubated for 1 hour. For biotinylated11C2, the plates were further incubated with 2 μg/ml HRP-conjugatedstreptavidin. For 55B, the plates are washed and incubated withHRP-conjugated donkey anti-rabbit IgG for 1 hour. At the end of theincubation, the wells were washed 6 times with 200 μl of PBS containing0.05% Tween-20, and the signal was visualized using 100 μl of thehorseradish peroxidase chromogenic substrate TMB (Kirkegaard & PerryLaboratories). The reaction was stopped with 100 μl of 1 M phosphoricacid, and the OD at 450 nm was measured. Non-specific binding wasdetermined in parallel incubations by omitting microtiter well coating.No signal was generated when WISP-1-Fc or a WISP-1 antibody was omitted.

Using the antibody 9C10 for capture and the antibodies 11C2 and 55B fordetection, an ELISA was conducted capable of detecting concentration ofWISP-1 as low as 0.4 μg/ml (FIG. 14). This ELISA may be useful fordetecting WISP-1 protein in biological fluids such as serum.

Example 14

Maxisorb plates were coated overnight at 4° C. with 50 μl/well of 10μg/ml heparin (Sigma). The non specific binding sites were blocked with200 μl of PBS/3% BSA for 1 hour. The plates were then incubated for 1hour with 50 μl of 6 μg/ml hWISP-1-Fc in PBS/3% BSA in the presence ofserial dilutions of WISP-1 antibodies. The plates were washed withPBS/0.05% Tween and further incubated 1 hour with 50 μl of 2 μg/ml HRPconjugated anti-human IgG-Fc in PBS/3% BSA. The plates were washed, and100 μl of HRP substrate (TMB) was added. The color development wasstopped with 100 μl of 1 M phosphoric acid and the OD at 450 nm wasmeasured.

The WISP-1 antibodies 11C2, 5D4 and 9C11 inhibited WISP-1 binding toheparin with an IC₅₀ of 1.9, 2.5 and 3.7 μg/ml, respectively (FIG. 15).The antibody 3D11 moderately reduced WISP-1 binding to heparin with amaximal inhibition of 62% at the highest concentration tested (40μg/ml). The antibody 9C10 did not attenuate WISP-1 heparin binding,showing an inhibition curve similar to the irrelevant antibody control.

These results demonstrate that antibodies recognizing the variableregion can inhibit WISP-1 binding to heparin. Because the two WISP-1antibodies recognizing domain 1 have little or no effect on WISP-1binding to heparin, it is presently believed that the domain 1 is lesslikely to participate in this interaction.

Example 15

Haptotaxis was measured using a modified Boyden chamber system. Theunderside of 8 μm porosity 24-well format PET membrane filters (BectonDickinson, Franklin Lakes, N.J.) were coated overnight at 4° C. with 50μl of protein (50 μg/ml) in PBS. Normal rat kidney cells (NRK; 5×10⁴/0.5ml HGDMEM/10% FBS) were added to the upper chamber, the lower chamberwas filled with the same media and the plates were incubated at 37° C.The next day, the upper chamber was wiped with a cotton swab, and thecells that migrated to the lower side of the insert were stained andcounted under a microscope. Triplicate sets of data were averaged foreach condition. In certain cases, the coated inserts were washed andtreated for an additional 2 hours at room temperature.

In a transwell assay, WISP-1-Fc coated on the filters' lower surfaceinduced the haptotactic migration of NRK cells (FIG. 16A). Coating of anirrelevant IgG chimeric protein (TNFR-Fc) or addition of WISP-1-Fc tothe lower chamber did not promote migration. In the presence of coatedWISP-1-Fc, five different WISP-1 antibodies (9C10, 11C2, 3D11, 9C11,5D4) markedly inhibited cell migration. In the absence of coatedWISP-1-Fc, these antibodies did not show any effect on cell migration.These results demonstrate that WISP-1 antibodies can modulate cellmigration in the presence of WISP-1. By blocking cell migration, WISP-1antibodies may play an important therapeutic role in preventing cancerprogression.

A summary of the characteristics and properties of the 3D11, 9C10, 11C2,5D4, and 9C11 antibodies discussed in the Examples above is provided inFIG. 16B.

Example 16

Nine-week-old Swiss nu/nu female mice were injected into the lateraltail vein with 100 μl of a suspension containing 2.5×10⁵ cells. Startingon the day of the inoculation, the mice were treated semiweekly byintraperitoneal injection (10 mg/kg) of WISP-1 antibodies (9C11, 11C2,5D4, 9C10, 3D11) or isotype control antibody. After 3 weeks, lungs wereperfused with 4% neutral buffered formalin, excised and H&E stainedsections were generated. Longitudinal sections of the left lung, and atransverse section of the cranial, medial, caudal, and accessory lobesof the right lung were evaluated. For each slide the number ofmetastatic foci was counted and the average area of metastatic foci wasmeasured using the SPOT RT software (Diagnostic InstrumentsIncorporated, Burlingame, Calif.). The area (μm²) was determined for atleast five individual metastatic foci in four sections of lung.

After 3 weeks, the severity of the lesions found in WISP-1 antibodytreated animals was greatly attenuated compared to control (FIG. 17 a,b). The number of nodules and the average area of the metastatic focifound in mice treated with WISP-1 antibodies (n=5) were reduced comparedto animals treated with a control antibody (FIG. 17 c,d). Furthermore,upon WISP-1 antibody treatment, the total pulmonary area covered by thelesions was reduced by 82-97% compared to animal treated with an isotypecontrol antibody (FIG. 17 e). These results demonstrate the in vivoefficacy of WISP-1 antibodies at reducing the tumor burden related tometastasis. Although the mechanism of action of the WISP-1 antibodies isnot fully understood, it is believed their efficacy may be mediated by agrowth reducing ability and/or a capacity at inhibiting the motility,invasion and seeding of cancer cells at a tissue site.

Example 17

Full length mouse WISP-1 (GenBank accession number NM_(—)018865) wascloned into the pRK mammalian expression vector. The resulting construct(pRK-WISP-1; 18 ug) was co-transfected with 2 ug pSVi-puromycin plasmidin a 4T1 mouse mammary adenocarcinoma cell line (obtained from Dr. FredMiller, Barbara Ann Karmanos Cancer Institute, Detroit, Mich.) usingFugene6 (Roche) according to manufacturer's instructions. After 2 days,cells were selected in 2 μg/ml puromycin. After 2 weeks, clones wereisolated and WISP-1 expression was evaluated by immunofluorescence. Thesame procedure was used to generate control cell lines using an emptyvector. The resulting 4T1/control and 4T1/WISP-1 cell lines weremaintained in Iscove's Media containing 10% FBS and 3 μg/ml puromycin.

WISP-1 expression in 4T1/control, 4T1/WISP-1L, 4T1/WISP-1H, NRK/control,NRK/WISP-1L and NRK/WISP-1H cells lines was measured bysemi-quantitative RT-PCR (Taqman) using primers and probes that do notdistinguish between human and mouse WISP-1 gene.

WISP-1 was not expressed in 4T1/control cell lines. WISP-1 expression in4T1/WISP-1H cell line was 2 fold higher than in 4T1/WISP-1L but 700 foldlower than in NRK/WISP-1H (FIG. 18).

Example 18

To evaluate WISP-1 effect on 4T1 cell scattering, 100,000 4T1/control,4T1/WISP-1L or 4T1/WISP-1H cells were seeded in 6 well plate in Iscove'sMedia containing 10% fetal bovine serum. When plated at a low density,4T1/control cells proliferated and formed well defined colonies (FIG.19). Proliferating 4T1/WISP-1L cells formed less defined groups as somecells departed from the growing colonies (FIG. 19). No 4T1/WISP-1Hcolonies were seen, and cells scattered in a random pattern (FIG. 19).Together these results suggest that WISP-1 expression promotes cellmigration.

Example 19

WISP-1 effect on 4T1 cell invasion was also evaluated using a Matrigelcoated modified Boyden chamber system of 8 μm porosity 24-well formatPET membrane filters (Falcon). 4T1/control, 4T1/WISP-1L or 4T1/WISP-1Hcells (100,000 cells) were added to the upper chamber in 0.5 ml Iscove'sMedia. The lower chamber was filled with 0.75 ml of the same mediacontaining 5% fetal bovine serum and the plates were incubated overnightat 37° C. The next day the upper chamber was wiped with a cotton swaband the cells that migrated to the lower side of the insert were stainedwith the Diff-Quik Stain Kit (Dade Behring Inc.) and counted under themicroscope. Triplicate sets of data were averaged for each condition.4T1/WISP-1H and 4T1/WISP-1L cells demonstrated a 12 and 6 fold increaseinvasion compared to 4T1/control, respectively (FIG. 20). These resultssuggest that WISP-1 promotes tumorigenic mammary epithelial cellinvasion and may play a role in metastasis.

Example 20

WISP-1 effects on mammary epithelial cells tumorigenesis were evaluatedby injecting 1.5×10⁵ cells (4T1/control 1, 4T1/control 2, 4T1/WISP-1L or4T1/WISP-1H) into the fourth mammary fat pad of 6-8 week old femaleBALB/c mice (6 mice/group). Tumor volumes were measured three times perweek. Thirty one days after injection, the mice were sacrificed, thetumors were excised and weighted.

Inoculation of 4T1/WISP-1L cells (FIG. 21 a; empty sqares) and4T1/WISP-1H cells (FIG. 21 a; filled sqares) generated faster growingtumors compared to 4T1/control 1 and 4T1/control 2 cells (FIG. 21 a;empty and filled circles). After 31 days, the tumors formed by4T1/WISP-1L and 4T1/WISP-1L inoculated were 4 fold larger (FIG. 21 a)and 3 fold heavier (FIG. 21 b) than the tumors formed by 4T1/controlcells. These results suggest that WISP-1 may increase proliferation ofmammary epithelial tumor cells.

Example 21

The expression of HAS2 and CD44 in tumors formed by the inoculated4T1/control, 4T1/WISP-1L and 4T1/WISP-1H cells was measured. Whencompared to 4T1/control cells tumor, CD44 expression was increasedbetween 5 and 23 fold in 4T1/WISP-1 cells tumors (FIG. 22). On the otherhand, HAS2 expression remained identical in all tumors analyzed. Theseresults suggest that WISP-1 increases expression of CD44 in miceinoculated with 4T1 cells. The overexpression of CD44 in these tumorsmay contribute to the promotion of metastasis.

Example 22

WISP-1 effects on mammary epithelial cells metastasis were evaluated byinoculating 4T1 cells in mice mammary fat pads (see Example 20) andexamining the extent of the metastatic propagation by micro computertomography and histology. After 31 days, the mice inoculated with4T1/WISP-1L or 4T1/WISP-1H cells had extensive lung metastasis (FIGS. 23b and 23 d) compared to the 4T1/control injected mice (FIGS. 23 a and 23c). No significant differences were seen between the metastaticpotential of 4T1/WISP-1L and 4T1/WISP-1H cells. The mice inoculated with4T1/control cells had an average of 2.11 pulmonary foci with an averagemass of 0.68 grams whereas mice inoculated with 4T1/WISP-1 cells had anaverage of 20.25 pulmonary foci with an average mass of 12.46 grams.Also, the average histology score for the lung of 4T1/WISP-1 cellsinjected mice was 0.92 compared to 0.11 for the NRK/control cellsinjected mice.

Using immunohistochemistry, it was also observed that the 4T1/WISP-1pulmonary metastatic foci expressed high levels of CD44 (FIG. 23 e). Inthese tumors, CD44 was localized at the plasma membrane of the4T1/WISP-1 cells (FIG. 23 f). Together these results demonstrate thatWISP-1 promotes the metastatic potential of 4T1 cells, increasing thenumber of pulmonary metastatic foci (10 fold) and size (18 fold). Also,CD44 increased expression was maintained after the 4T1/WISP-1 hadmetastasized to the lungs.

Clinical observations of mice inoculated with 4T1/control 1, or4T1/WISP-1 cells demonstrated that WISP-1 expression promoted metastasisat additional secondary sites. Two mice inoculated with 4T1/WISP-1H hada discolored white mass on the kidney whereas no other mice had evidenceof kidney tumors.

Example 23

The human WISP-1 full length and deletion mutant constructs describedabove were transfected in normal rat kidney cells (NRK-49F). After 48hours, the culture media was supplemented with 2 μg/ml puromycin. After2 weeks, the pools of expressing cells were analyzed and the resultingcell lines were maintained in High glucose Dulbecco's Modified Eagle'sMedium (HGDMEM) supplemented with 10% fetal calf serum (FBS) and 2 μg/mlpuromycin.

WISP-1 expression in NRK/control, NRK/WISP-1_(—)1234, NRK/WISP-1_(—)134,NRK/WISP-1_(—)234, NRK/WISP-1_(—)123 and NRK/WISP-1_(—)124 cells lineswas measured by semi-quantitative RT-PCR (Taqman). Results are expressedas fold relative to WISP-1 expression in NRK/WISP-1_(—)1234 (FIG. 24A).WISP-1 expression in NRK/WISP-1_(—)134 and NRK/WISP-1_(—)234 is 0.035and 0.09 fold respectively compared to NRK/WISP-1_(—)1234.NRK/WISP-1_(—)123 expresses 16.1 fold and NRK/WISP-1_(—)124 7.6 foldcompared to NRK/WISP-1_(—)1234.

The contribution of particular domains to WISP-1 promoted tumorigenesiswas evaluated by injecting 5×10⁶ cells (NRK/control, NRK/WISP-1_(—)1234,NRK/WISP-1_(—)123, NRK/WISP-1_(—)124, NRK/WISP-1_(—)134,NRK/WISP-1_(—)234 cells) subcutaneously in 6-8 week old female nude mice(10 mice/group). Tumor volumes were measured weekly. After 35 days themice were sacrificed, the tumors were excised and analyzedhistologically.

Inoculation of NRK/WISP-1_(—)1234 (FIG. 24B; filled triangles),NRK/WISP-1_(—)134 (FIG. 24B; filled circles) and NRK/WISP-1_(—)234 cells(FIG. 24B; filled squares) generated tumors. However the inoculation ofNRK/WISP-1_(—)123 (FIG. 24B; empty squares), NRK/WISP-1_(—)124 (FIG.24B; empty circles) and NRK/control (FIG. 24B; empty triangles) did notgenerate tumors. After 35 days, the tumors formed by NRK/WISP-1_(—)234cells were comparable in size to the tumors formed by NRK/WISP-1_(—)1234cells. However the tumors formed by NRK/WISP-1_(—)134 cells were 2.5fold larger than the tumors formed by NRK/WISP-1_(—)1234 cells. Theseresults suggest that the domains 1 and 2 are not required for WISP-1promoted NRK tumorigenesis but that the domains 3 and 4 are required forWISP-1 promoted NRK tumorigenesis. It is believed that WISP-1antagonists of the invention may preferably bind to and/or block domains3 and/or 4 of WISP-1.

In addition, results suggest the deletion of the domain 2 of WISP-1 mayincrease its tumorigenic potential.

Histological analysis of the excised tumors (FIG. 25) revealed that theneoplastic cells from NRK/WISP-1_(—)234 tumors were phenotypicallysimilar to the neoplastic cells from NRK/WISP-1_(—)1234 tumors. In thesetumors, cells appeared fibroblastic, differentiated and spindloid.However the neoplastic cells from NRK/WISP-1_(—)134 tumors werephenotypically different and appeared less differentiated, frequentlymultinucleated and had a brisk mitotic rate as demonstrated by thepresence of multiple mitotic figures (arrows). In addition, neoplasticcells invaded several blood vessels adjacent to NRK/WISP-1_(—)134 tumors(FIG. 26). These results indicate that WISP-1_(—)134 expression mayconfer tumorigenicity to NRK cells by promoting their in vivo growth andinvasive potential. Moreover the expression of this construct induces aphenotypic transformation characteristic of aggressive neoplastic cells.

Example 24

The contribution of the particular WISP-1 domains to the promoted 4T1cell invasion was evaluated using a Matrigel coated modified Boydenchamber system of 8 μm porosity 24-well format PET membrane filters (BDBiosciences). HEK 293 cells (20×10⁶) stably expressing the previouslymentioned WISP-1 constructs (WISP-1_(—)1234, WISP-1_(—)134,WISP-1_(—)123, WISP-1_(—)12, WISP-1_(—)23, WISP-1_(—)3, WISP-1_(—)1 andWISP-1_(—)4) were seeded in the lower chamber with in 0.75 ml of 50:50Media (1:1 mixture of DME and Ham's F-12 medium) containing 5% fetalbovine serum and the plates were kept overnight at 37° C. The next day,4T1 cells (100,000 cells) were added to the upper chamber in 0.5 ml ofserum free 50:50 media and the plates were incubated overnight at 37° C.The following day the upper chamber was wiped with a cotton swab and thecells that migrated to the lower side of the insert were stained withthe Diff-Quik Stain Kit (Dade Behring Inc.) and counted under themicroscope. Triplicate sets of data were averaged for each condition andresults are expressed as fold invasion compared to a control in whichHEK 293 cells transfected with an empty vector were plated in the lowerchamber. 4T1 cells demonstrated a 4 fold increase invasion when HEK 293cells expressing constructs WISP-1_(—)1234, WISP-1_(—)134 or WISP-123were seeded in the lower chamber (FIG. 27). HEK 293 cells expressing theconstruct WISP-1_(—)12 increased 4T1 cells invasion by only 2.5 fold.The increase in 4T1 cell invasion was marginal when HEK 293 cellsexpressing WISP-1_(—)23, WISP-1_(—)3, WISP-1_(—)1 or WISP-1_(—)4 wereplated in the lower chamber. These results suggest that the domain 1 isrequired for WISP-1 promoted 4T1 cell invasion. In addition, the datashows that WISP-1's domain 1 alone is likely not sufficient to promote4T1 cell invasion.

Example 25

An assay was conducted to evaluate the antagonistic activity of WISP-1'sdomain 1 on WISP-1 promoted 4T1 cell invasion. The domain 1 of WISP-1was cloned into an expression vector immediately upstream of the humanIgG₁ Fc region sequence. The resulting recombinant fusion protein(WISP-1-domain 1-Fc) was synthesized in a mammalian expression systemusing Chinese hamster ovary (CHO) cells and purified to homogeneity fromconditioned medium by affinity chromatography on a Protein A-Sepharose 4Fast Flow (Amersham Pharmacia Biotech). WISP-1's domain 1 was alsoexpressed with an amino terminal hexa-histidine tag(WISP-1-domain-1-His) in a CHO expression system and purified tohomogeneity from culture supernatant by affinity chromatography on aNi²⁺-NTA agarose column (Qiagen). Fractions containing the elutedWISP-1-domain-1-His were then pooled and dialyzed.

The antagonistic effect of purified soluble WISP-1-domain-1 on WISP-1promoted 4T1 cell invasion was evaluated using a Matrigel coatedmodified Boyden chamber system of 8 μm porosity 24-well format PETmembrane filters (BD Biosciences). 4T1/control or 4T1/WISP-1 cells(100,000 cells) were added to the upper chamber in 0.5 ml Iscove'sMedia. The lower chamber was filled with 0.75 ml of the same mediacontaining 5% fetal bovine serum. Various concentrations ofWISP-1-domain-1-Fc or WISP-1-domain-1-His were added to the upper andlower chambers and the plates were incubated overnight at 37° C. Thenext day the upper chamber was wiped with a cotton swab and the cellsthat migrated to the lower side of the insert were stained with theDiff-Quik Stain Kit (Dade Behring Inc.) and counted under themicroscope. Triplicate sets of data were averaged for each condition.The results are expressed as relative fold invasion compared to4T1/control cells. WISP-1-domain-1-His and WISP-1-domain-1-Fcdemonstrated a dose dependent antagonistic activity toward WISP-1promoted 4T1 invasion (FIG. 28). Both WISP-1-domain-1-His andWISP-1-domain-1-Fc showed an apparent IC₅₀ of 0.01 μg/ml. These resultssuggest that purified WISP-1-domain-1 antagonizes WISP-1 promoted cellinvasion.

Example 26

An animal study was conducted to evaluate WISP-1 expression in the HPACxenograft model of human pancreatic adenocarcinomas. 5×10⁶ humanpancreatic adenocarcinomas cells (HPAC cell line, cat. no. CRL-2119;ATCC, Manassas, Va., USA;) were injected subcutaneously in 6-8 week oldfemale nude mice. After 21 days the mice were sacrificed, the tumorswere excised and WISP-1 expression was analyzed by in situ hybridizationusing a human WISP-1 specific probe and a mouse WISP-1 specific probe(FIG. 29). The results demonstrate a strong mouse WISP-1 expression inareas corresponding to tumor stroma. On the other hand, no human WISP-1expression was found in the excised tumors. In addition, the tumorsrevealed a strong α-smooth muscle actin and a weak vimentinimmunohistochemical staining over the tumoral stroma. These resultsindicate that HPAC cells inoculation in nude mice induces therecruitment of a mouse myofibroblastic stroma in which WISP-1 isexpressed.

Example 27

An animal study was conducted to evaluate the in vivo efficacy ofWISP-1-domain-1 at retarding tumor growth. The human WISP-1_(—)1,WISP-1_(—)12 constructs cloned into an expression vector (pIRES puro2;Clontech Laboratories, Palo Alto, Calif.) were transfected in the HPAChuman pancreatic adenocarcinomas cell line. After 48 hours, the culturemedia was supplemented with 2 μg/ml puromycin. After 2 weeks, the poolsof expressing cells were analyzed and the resulting cell lines weremaintained in 50:50 media (1:1 mixture of DME and Ham's F-12 medium)supplemented with 10% fetal calf serum (FBS) and 2 μg/ml puromycin.

The in vivo efficacy of WISP-1-domain-1 at retarding tumor growth wasevaluated by injecting 5×10⁶ cells (HPAC/WISP-1_(—)12, HPAC/WISP-1_(—)1cells) subcutaneously in 6-8 week old female nude mice (10 mice/group).Tumor volumes were measured twice weekly until day 25. The resultsdemonstrate that the expression of WISP-1_(—)1 or WISP-1_(—)12 in HPACcells retards their in vivo growth. The tumor volume ofHPAC/WISP-1_(—)12 and HPAC/WISP-1_(—)1 cells were 12% and 54% smallerrespectively compared to HPAC control cells tumors (FIG. 30). Theseresults indicates that WISP-1-domain-1 and WISP-1-domain-12 expressed byHPAC cells retard their tumor growth possibly by antagonizing mouseWISP-1 tumor promoting activities.

Example 28

In situ hybridization studies were conducted to evaluate WISP-1expression in primary human pancreatic adenocarcinomas. Using a tumormicroarray (TMA), it was shown that 68% ( 125/184) of the samples coreswere positive for WISP-1 expression. In addition, in these samplesWISP-1 expression was restricted to the stromal compartment (FIG. 31).These results indicate a high incidence of WISP-1 expression in thestromal compartment of human primary pancreatic tumors.

Example 29

Immunohistochemical staining was conducted to evaluate WISP-1 expressionin primary colon adenocarcinomas. A panel of 15 samples of primary humanadenocarcinomas were stained for WISP-1 using a WISP-1 specificpolyclonal goat antibody (R&D systems; cat. No. AF1627). WISP-1 wasdetected in 100% ( 15/15) of the samples analyzed. In all cases WISP-1was located in the stromal compartment surrounding the tumors (FIG. 32).These results indicate that WISP-1 is prevalent in the stromalcompartment of human colon adenocarcinomas.

WISP-1 Constructs: Domain 2,3: (SEQ ID NO:12)GAATTCACCATGAGGTGGTTCCTGCCCTGGACGCTGGCAGCAGTGACAGCAGCAGCCGCCAGCACCGTCCTGGCCACTGCAGTGGTCGGTGTGGGCTGCGTCCTGGATGGGGTGCGCTACAACAACGGCCAGTCCTTCCAGCCTAACTGCAAGTACAACTGCACGTGCATCGACGGCGCGGTGGGCTGCACACCACTGTGCCTCCGAGTGCGCCCCCCGCGTCTCTGGTGCCCCCACCCGCGGCGCGTGAGCATACCTGGCCACTGCTGTGAGCAGTGGGTATGTGAGGACGACGCCAAGAGGCCACGCAAGACCGCACCCCGTGACACAGCAGCCTTCGATGCTGTGGGTGAGGTGGAGGCATGGCACAGGAACTGCATAGCCTACACAAGCCCCTGGAGCCCTTGCTCCACCAGCTGCGGCCTGGGGGTCTCCACTCGGATCTCCAATGTTAACGCCCAGTGCTGGCCTGAGCAAGAGAGCCGCCTCTGCAACTTGCGGCCATGCGATGTGGACATCCATACACTCATTAAGGCGGCCGCACACCACCATCACCATCACCATCACTAAGTGAGGCCGCATAGATAACTGATCCAGTGTGCTGGAATTAATTCGCTGTCTGCGA Domain 1-IgG: (SEQ ID NO:13)      CCTCGGTTCTATCGATTGAATTCCACCATGAGGTGGTTCCTGCCCTGGACGCTGGCAGCAGTGACAGCAGCAGCCGCCAGCACCGTCCTGGCCACGGCCCTCTCTCCAGCCCCTACGACCATGGACTTTACTCCAGCTCCACTGGAGGACACCTCCTCACGCCCCCAATTCTGCAAGTGGCCATGTGAGTGCCCGCCATCCCCACCCCGCTGCCCGCTGGGGGTCAGCCTCATCACAGATGGCTGTGAGTGCTGTAAGATGTGCGCTCAGCAGCTTGGGGACAACTGCACGGAGGCTGCCATCTGTGACCCCCACCGGGGCCTCTACTGTGACTACAGCGGGGACCGCCCGAGGTACGCAATAGGAGTGTGTGCACAGGTGGTCGGTGTGGGGGTCACCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAACGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAAGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCCTAGAGTCGACCTGCAGAAGCTTGGCCGCCATGGCCCDeposit of Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209,USA (ATCC): Material ATCC Dep. No. Deposit Date 3D11.D7 PTA-4624 Sep. 4,2002 11C2.C10 PTA-4628 Sep. 4, 2002 9C10.F5 PTA-4626 Sep. 4, 2002 5D4.F6PTA-4625 Sep. 4, 2002 9C11.C7 PTA-4627 Sep. 4, 2002

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC '122 and the Commissioner's rules pursuantthereto (including 37 CFR '1.14 with particular reference to 886 OG638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The foregoing written description is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the example presented herein.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and fall within the scope of theappended claims.

1. An isolated WISP-1 antagonist which inhibits or neutralizes inductionor secretion of HAS2, HA, CD44 or RHAMM by native WISP-1 polypeptide inat least one type of mammalian cell, said antagonist being selected fromthe group consisting of an anti-WISP-1 antibody, a WISP-1 immunoadhesinand a WISP-1 variant.
 2. The antagonist of claim 1, wherein saidantagonist comprises an anti-WISP-1 antibody.
 3. The antagonist of claim2, wherein said anti-WISP-1 antibody binds native human WISP-1polypeptide comprising amino acids 23-367 of FIGS. 9A-9C (SEQ ID NO:1)or one or more domains of WISP-1 polypeptide comprising amino acidsencoded by the sequences of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10 or
 11. 4.The antagonist of claim 2 or 3, wherein said anti-WISP-1 antibody is achimeric, humanized or human antibody.
 5. The antagonist of claim 1,wherein said antagonist comprises a WISP-1 immunoadhesin.
 6. Theantagonist of claim 5, wherein said antagonist comprises a human WISP-1sequence fused to a Fc region of an immunoglobulin.
 7. A compositioncomprising the antagonist of any of claims 1-6 and a carrier.
 8. Thecomposition of claim 7 wherein said carrier is apharmaceutically-acceptable carrier.
 9. A method of inhibiting orneutralizing WISP-1 induction or secretion of HAS2, HA, CD44 or RHAMM inmammalian cells, comprising exposing said mammalian cells to aneffective amount of WISP-1 antagonist, wherein said WISP-1 antagonist isselected from the group consisting of d) a WISP-1 immunoadhesin; e) aWISP-1 polypeptide linked to a nonproteinaceous polymer selected fromthe group consisting of polyethylene glycol, polypropylene glycol, andpolyoxyalkylene; f) a WISP-1 antibody; and d) a WISP-1 variant.
 10. Themethod of claim 9, wherein said WISP-1 immunoadhesin comprises a WISP-1sequence fused to a Fc region of an immunoglobulin.
 11. The method ofclaim 9, wherein said anti-WISP-1 antibody binds native human WISP-1comprising amino acids 23-367 of FIGS. 9A-9C (SEQ ID NO:1) or one ormore domains of WISP-1 polypeptide comprising amino acids encoded by thesequences of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10 or
 12. The method ofclaim 9 or 11, wherein said anti-WISP-1 antibody is a chimeric,humanized or human antibody.
 13. The method of claim 9, wherein saidmammalian cells comprise cancer cells.
 14. The method of claim 13,wherein said mammalian cells comprise colon or colorectal cancer cells,breast cancer cells, lung cancer cells or brain cancer cells.
 15. Amethod of treating cancer in a mammal, comprising administering to saidmammal an effective amount of WISP-1 antagonist, wherein said antagonistis selected from the group consisting of an anti-WISP-1 antibody, aWISP-1 immunoadhesin and a WISP-1 variant.
 16. The method of claim 15,wherein said cancer comprises colon or colorectal cancer cells, breastcancer cells, lung cancer cells, or brain cancer cells.
 17. The methodof claim 15, wherein said antagonist inhibits or reduces cancer cellgrowth or metastasis.
 18. The method of claim 15, wherein saidanti-WISP-1 antibody binds native human WISP-1 comprising amino acids23-367 of FIGS. 9A-9C (SEQ ID NO:1) or one or more domains of WISP-1polypeptide comprising amino acids encoded by the sequences of SEQ IDNO:3, 4, 5, 6, 7, 8, 9, 10 or
 19. The method of claim 15 or 18 whereinsaid anti-WISP-1 antibody is a chimeric, humanized or human antibody.20. The method of claim 15, wherein chemotherapy, radiation, prodrug,cytotoxic agent, growth inhibitory agent, or cytokine is alsoadministered to said mammal.
 21. The method of claim 15 wherein saidantagonist wherein said antagonist inhibits or neutralizes induction orsecretion of HAS2, HA, CD44 or RHAMM by native human WISP-1 polypeptidein at least one type of mammalian cell.
 22. A monoclonal antibodycomprising the 3D11, 11C2, 9C10, 5D4, or 9C11 antibody secreted by thehybridoma deposited with ATCC as accession number PTA-4624, PTA-4628,PTA-4626, PTA-4625, or PTA-4627, respectively.
 23. A monoclonal antibodywhich binds to the same epitope as the epitope to which the 3D11, 11C2,9C10, 5D4, or 9C11 monoclonal antibody produced by the hybridoma cellline deposited as ATCC accession number PTA-4624, PTA-4628, PTA-4626,PTA-4625, or PTA-4627, respectively, binds.
 24. The hybridoma cell linewhich produces monoclonal antibody 3D11, 11C2, 9C10, 5D4, or 9C11produced by the hybridoma cell line deposited as ATCC accession numberPTA-4624, PTA-4628, PTA-4626, PTA-4625, or PTA-4627, respectively. 25.An isolated anti-WISP-1 antibody, comprising an antibody which binds toWISP-1 polypeptide and competitively inhibits binding of the monoclonalantibody 3D11, 11C2, 9C10, 5D4, or 9C11 produced by the hybridoma cellline deposited as ATCC accession number PTA-4624, PTA-4628, PTA-4626,PTA-4625, or PTA-4627, respectively, to said WISP-1 polypeptide.
 26. Theantibody of claim 25 which is a chimeric, human or humanized antibody.27. A chimeric anti-WISP-1 antibody which specifically binds to WISP-1polypeptide and said antibody comprises a sequence derived from the3D11, 11C2, 9C10, 5D4, or 9C11 monoclonal antibody produced by thehybridoma cell line deposited as ATCC accession number PTA-4624,PTA-4628, PTA-4626, PTA-4625, or PTA-4627, respectively.
 28. Theantibody of claim 27 wherein said derived sequence is the variable orhypervariable region of the 3D11, 11C2, 9C10, 5D4, or 9C11 monoclonalantibody.
 29. The WISP-1 antagonist or antibody of any of the precedingclaims wherein said antibody is an anti-WISP-1 antibody linked to one ormore agents selected from the group consisting of non-proteinaceouspolymer, cytotoxic agent, enzyme, radioisotope, fluorescent compound,and chemiluminescent compound.
 30. The method of claim 16, wherein saidantagonist inhibits or reduces lung cancer cell metastasis at a site inthe mammal secondary or different from the primary lung tumor site inthe mammal.